Engineering synthetic brain penetrating gene vectors

ABSTRACT

A synthetic gene delivery platform with a dense surface coating of hydrophilic and neutrally charged PEG, capable of rapid diffusion and widespread distribution in brain tissue, and highly effective gene delivery to target cells therein has been developed. Nanoparticles including nucleic acids, are formed of a blend of biocompatible hydrophilic cationic polymers and they hydrophilic cationic polymer conjugated to hydrophilic neutrally charged polymers such as polyethylene glycol. The nanoparticles are coated with polyethylene glycol at a density that imparts a near neutral charge and optimizes rapid diffusion through the brain parenchyma. Methods of treating a disease or disorder of the brain including administering a therapeutically effective amount of nanoparticles densely coated with polyethylene glycol are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/310,535 filed Nov. 11, 2016, which is a 371 application ofInternational Application No. PCT/US2015/030381, filed May 12, 2015,which claims priority to and the benefit of U.S. Ser. No. 61/991,898filed May 12, 2014, U.S. Ser. No. 61/991,920 filed May 12, 2014 and U.S.Ser. No. 62/001,994 filed May 22, 2014, the contents of which areexpressly incorporated hereby by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under EB003558 andCA164789 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention is generally in the field of gene delivery, and inparticular, in delivering nucleic acids across biological barriers usingcoated particles to penetrate the brain parenchyma and achieve highlevel widespread transgene expression.

BACKGROUND OF THE INVENTION

Patients with neurological diseases, including Parkinson's disease,Alzheimer's disease, brain tumors and most neurogenetic disorders,suffer from severe debilitating symptoms and lack of therapeutic optionsthat provide curative treatment. The accumulated knowledge of specificgenetic targets that can alter or reverse the natural history of centralnervous system (CNS) diseases has rendered gene therapy an attractivetherapeutic strategy [O'Mahony, A. M., et al., J Pharm Sci, 2013.102(10): 3469-3484; Lentz, et al., Neurobiol Dis, 2012. 48(2):179-188.]. Multiple preclinical and clinical studies have aimed toimprove the delivery of nucleic acids to the CNS using leading viral ornon-viral gene vectors with specific focus to enhancing the level anddistribution of transgene expression throughout the brain tissue[O'Mahony, et al., J Pharm Sci, 2013. 102(10): 3469-3484;Perez-Martinez, et al., J Alzheimers Dis, 2012. 31(4): 697-710].

Viral gene vectors, though relatively efficient, have been limited byone or more drawbacks, including low packaging capacity, technicaldifficulties in scale-up, high cost of production [Thomas, et al., NatRev Genet, 2003. 4(5): 346-358.] and risk of mutagenesis [Olsen andStein, N Engl J Med, 2004. 350(21): 2167-2179.]. Furthermore, despitethe immune privileged nature of the CNS, neutralizing immune responsesmay occur secondary to repeated administrations or prior exposures[Lentz, et al., Neurobiol Dis, 2012. 48(2): 179-188; Xiao, X., et al., JVirol, 1996. 70(11): 8098-8108; Chirmule, N., et al., J Virol, 2000.74(5): 2420-2425; Lowenstein, P. R., et al., Curr Gene Ther, 2007. 7(5):p. 347-60; Lowenstein, P. R., et al., Neurotherapeutics, 2007. 4(4):715-724; Voges, J., et al., Ann Neurol, 2003. 54(4): 479-487.].

Non-viral gene vectors can offer an attractive alternate strategy forgene delivery without many of these limitations [O'Mahony, A. M., etal., J Pharm Sci, 2013. 102(10): 3469-3484]. Cationic polymer-based genevectors provide a tailorable platform for DNA condensation and efficientgene transfer in vitro and in vivo. Their positive charge density allowsfor stable compaction of negatively charged nucleic acids [Sun, X. andN. Zhang, Mini Rev Med Chem, 2010, 10(2): 108-125; Dunlap, D. D., etal., Nucleic Acids Res, 1997. 25(15): 3095-3101] and protects them fromenzymatic degradation [Kukowska-Latallo, J. F., et al., Hum Gene Ther,2000. 11(10): 1385-1395.]. Also, the number of protonable aminesprovides increased buffering capacity that facilitates endosome escapevia the “proton sponge effect”, leading to efficient transfection[Akinc, A., et al., J Gene Med, 2005. 7(5): 657-663]. A wide variety ofcationic polymers have been developed for this purpose, offering genevectors with diverse physicochemical profiles and in vivo behaviors[Mintzer, M. A. and E. E. Simanek. Chem Rev, 2009. 109(2): 259-302;Pathak, et al., Biotechnol J, 2009. 4(11): 1559-72.].

However, non-viral gene vectors still face a number of barriers prior toreaching the target cells in the brain [O'Mahony, et al., J Pharm Sci,2013. 102(10): 3469-3484]. Various strategies have been developed tomanipulate or bypass the blood brain barrier (BBB) [Jain, Nanomedicine(Lond), 2012. 7(8): 1225-33; Wohlfart, et al., J Control Release, 2012.161(2): 264-273.], which is the primary barrier to the systemic deliveryof gene vectors to the brain. These approaches include, but are notlimited to, direct, local administration to the CNS [Patel, et al.,Advanced Drug Delivery Reviews, 2012. 64(7):701-705] and reversibledisruption of the BBB via focused ultrasound [Vykhodtseva, et al.,Ultrasonics, 2008. 48(4): 279-296] or chemical reagents [Kroll, et al.,Neurosurgery, 1998. 42(5): 1083-1099; discussion 1099-100.]. However,once beyond the BBB, the anisotropic and electrostatically chargedextracellular matrix (ECM) found between brain cells has been widelyrecognized as another critical barrier [Nance, et al., Sci Transl Med,2012. 4(149): 149ra119; Sykova, et al., Physiol Rev, 2008. 88(4):1277-1340; Zamecnik, J., Acta Neuropathol, 2005. 110(5):435-442]. This‘brain tissue barrier’, regardless of administration method, hamperswidespread distribution of macromolecules and nanoparticles in thebrain, thereby limiting their coverage throughout the disseminatedtarget area of neurological diseases [Voges, J., et al., Ann Neurol,2003. 54(4): 479-487; Nance, E. A., et al., Sci Transl Med, 2012.4(149): 149ra119; Sykova, et al., Physiol Rev, 2008. 88(4): 1277-340;MacKay, et al., Brain Res, 2005. 1035(2): 139-153]. The ECM is rich inhyaluronan, chondroitin sulfate, proteoglycans, link proteins andtenascins and may provide a negatively charged adhesive barrier to thepenetration of cationic polymeric gene vectors [Sykova, et al., PhysiolRev, 2008. 88(4): 1277-1340; Zimmermann, et al., Histochem Cell Biol,2008. 130(4): 635-653]. Moreover, the pore size of the ECM imposes asteric barrier for the movement of nanoparticles in the CNS withnon-adhesive 114 nm, but not 200 nm, particles able to penetrate thebrain tissue [Nance, E. A., et al., Sci Transl Med, 2012. 4(149): p.149ra119; Kenny, G. D., et al., Biomaterials, 2013. 34(36): 9190-9200.It has been shown that sub-100 nm nanoparticles exceptionallywell-coated with hydrophilic and neutrally charged polyethylene glycol(PEG) rapidly diffuse in the brain ECM allowing the Widespreaddistribution of therapeutics [Nance, E. A., et al., Sci Transl Med,2012. 4(149): p. 149ra119].

Convection enhanced delivery (CED) can be applied to further enhance thedistribution of therapeutics by providing a pressure gradient duringintracranial administration [Allard, et al., Biomaterials, 2009. 30(12):2302-2318.]. However, CED is unlikely to provide a significant benefitif particles remain entrapped in the brain parenchyma due to adhesiveinteractions and/or steric obstruction. Thus, physicochemical propertiesof particles that allow unhindered diffusion in the brain parenchymaremain critical for achieving enhanced particle penetration followingCED [Allard, et al., Biomaterials, 2009. 30(12): 2302-18; Kenny, et al.,Biomaterials, 2013. 34(36): 9190-9200]. However, even following CED, theinteractions between positively charged gene vectors and the negativelycharged ECM, confine cationic nanoparticles to the point of injectionand perivascular spaces, and limit their penetration into the brainparenchyma [MacKay, et al., Brain Res, 2005. 1035(2): 139-153; Kenny, etal., Biomaterials, 2013. 34(36): 9190-9200; Writer, et al., J ControlRelease, 2012. 162(2): p. 340-8.].

It is therefore an object of the present invention to provide optimizedphysicochemical properties of stable gene vectors that can penetrate thebrain parenchyma thus improving distribution and transgene expressionthroughout the brain tissue.

It is a further object of the present invention to provide gene deliveryvectors with a favorable safety profile.

It is a yet further object of the present invention to combinenanoparticles with delivery strategies that can further enhance theirdistribution and transgene expression in the tissue, especially thebrain.

SUMMARY OF THE INVENTION

A synthetic gene delivery platform with a dense surface coating ofhydrophilic and neutrally charged hydrophilic polymer such aspolyethylene glycol (“PEG”) or polaxamer (polyethylene glycol orpolyalkylene oxid copolymers, PLURONIC®), capable of rapid diffusion andwidespread distribution in brain tissue, and highly effective genedelivery to target cells therein has been developed. Examplesdemonstrate densely PEGylated gene vectors, formulated from a mixture ofcationic polymers, such as 25 kDa polyethylenimine (PEI) or poly-Llysine (PLL), and the hydrophilic polymers conjugated to polymers suchas PEG, rapidly penetrate the brain parenchyma. Convection enhanceddelivery (CED) of these brain penetrating gene vectors provides anenhanced distribution of the gene vectors, resulting in highly effectivetransgene expression throughout the brain tissue.

The nanoparticles are formed from a blend of free polymer and polymerconjugated to a hydrophilic polymer such as polyethylene glycol. Theblending technique significantly improves nanoparticle compaction andincreases colloidal stability in high ionic strength solutions,including artificial cerebrospinal fluid. This strategy has been furtherapplied to develop peptide and biodegradable polymer based brainpenetrating gene vectors with similar attributes. As described in theexamples, nanoparticles including nucleic acid, a biocompatible,biodegradable cationic polymer and polyethylene glycol, wherein between90% and 75% of the cationic polymer are conjugated to polyethyleneglycol, and the nucleic acids are encapsulated within and/or areassociated with the surface of the nanoparticles. The nanoparticles arecoated with polyethylene glycol at a density that imparts a near neutralcharge and optimizes rapid diffusion through the brain parenchyma. Thenanoparticles preferably have a diameter of less than 114 nm, forexample, 50 nm.

In some embodiments the cationic polymer or polyethylene glycol isbranched. Branching enhances the amount of polyethylene glycolconjugated to the polymer, for example, by at least three times as muchpolyethylene glycol, as compared to conjugation of non-branchedpolyethylene glycol and non-branched polymer. In some embodiments thepolyethylene glycol has a molecular weight between 1,000 Daltons and10,000 Daltons, such as 5,000 Daltons.

In certain embodiments the cationic polymer is branchedpolyethyleneimine with a molecular weight between 10,000 Daltons and50,000 Daltons, for example 25,000 Daltons. In further embodiments themolar ratio of polyethylene glycol to polyethyleneimine is more than 8,preferably 26. In a particular embodiment 25% of the total amines derivefrom unconjugated branched polyethyleneimine. In other embodiments, thecationic polymer is poly-L lysine and the polyethylene glycol isbranched polyethylene glycol with a molecular weight of 5,000 Daltons.In one embodiment, 90% of the total poly-L lysine is conjugated withpolyethylene glycol.

Dosage formulations for the delivery of a therapeutic or prophylacticnucleic acid to the brain are also disclosed. The formulations include atherapeutically effective amount of nanoparticles densely coated withpolyethylene and a pharmaceutically acceptable excipient for deliveryinto the brain. The nanoparticles can be formulated for direct orindirect injection into the brain.

Methods of making nanoparticles densely coated with polyethylene glycolfor the delivery of nucleic acids to the brain are also provided. Themethods include preparing a blended polymer by mixing free polymer withpolymer conjugated to a polymer such as polyethylene glycol, adding thenucleic acid to the blended polymer and purifying the nanoparticles.

Methods for treating a disease or disorder of the brain, includingadministering to the brain a formulation including a therapeuticallyeffective amount of the nanoparticles to alleviate one or more symptomsof a disease or disorder of the brain and a pharmaceutically acceptableexcipient for delivery into the brain are also provided. The formulationcan be administered directly or indirectly to the brain. In someembodiments the formulation is administered systemically and thenanoparticles penetrate the brain by passing through the blood-brainbarrier. The particles can be administered in combination with one ormore techniques to bypass the blood brain barrier. Exemplary techniquesinclude convection enhanced delivery (CED), electron paramagneticresonance, ultrasound, and ultrasound application with microbubbles.Other methods of increasing uptake into the brain include formulatingwith an osmotic agent such as mannitol. The methods can be useful totreat one or more symptoms of a disease or disorder including, but notlimited to, tumors, neurological disorders, and brain injury or trauma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the hydrodynamic diameter of vectors (nm) overtime (hours) for UPN (▪), CPN (+) and BPN (♦), respectively. Size wasmeasured by dynamic light scattering (DLS) in aCSF at pH 7.0.Measurements continued for 24 hours every half an hour or untilpolydispersity (PDI)>0.5. Data represents the mean±SEM.

FIGS. 2A to 2D are bar graphs. FIGS. 2A-2C show the % cell viability ofrabbit primary astrocytes (FIG. 2A), rat primary astrocytes (FIG. 2B)and 9 L rat gliosarcoma cells (FIG. 2C), respectively, treated withvarious concentrations (1 mg/ml, 5 mg/ml or 10 mg/ml) of PEInanoparticles (UPN, CPN and BPN) or PEG-PLL nanoparticles, respectively.Cell viability was measured after 24 hr of treatment and compared tonon-treated controls. Data represents the mean±SEM. * Denotesstatistically significant (p<0.05) difference from 100%. FIG. 2D showshistopathology (inflammation and hemorrhage), scored using a customscale (0: no inflammation or hemorrhage; 1: mild; 2: moderate; 3:severe) for each of the PEI nanoparticles (UPN, CPN and BPN) or salinecontrol (Ctrl), respectively.

FIGS. 3A to 3D are bar graphs. FIGS. 3A and 3B show the % cell uptake of9 L rat gliosarcoma cells (FIG. 3A) and rat primary astrocytes (FIG.3B), treated with PEI nanoparticles (UPN, CPN and BPN), PEG-PLLnanoparticles, or saline control (Ctrl), respectively. FIGS. 3C and 3Dshow luciferase activity (RLU)/mg protein following in vitrotransfection of luciferase gene into 9 L rat gliosarcoma cells (FIG. 3C)and rat primary astrocytes (FIG. 3D), treated with PEI nanoparticles(UPN, CPN and BPN), PEG-PLL nanoparticles, or saline control (Ctrl),respectively. Data represents the mean±SEM. * Denotes statisticalsignificance P<0.05.

FIG. 4A shows trajectories of UPN, CPN and BPN nanoparticles over 20seconds, at a time scale of 1 second. The scale bar represents 0.25 μm.FIG. 4B is a graph showing ensemble-averaged geometric mean of meansquare displacements (MSDs) of PEI based gene vectors (UPN, CPN and BPN)as a function of time (seconds). Data represent the ensemble average ofat least three independent experiments, with N≥500 particles tracked foreach experiment. FIG. 4C is a panel of histograms showing % of genevectors over log mean square displacements (MSD) for each respectivegene vector (UPN, CPN and BPN) from at least three independentexperiments at a timescale of τ=1 sec.

FIG. 5A is a graph showing image based Matlab quantification of area ofdistribution of PEI-based nanoparticles (mm²) as a function of distance(mm) from the injection site, for CPN (∘) and BPN (●) (N=6). FIG. 5B isa histogram showing the volume of distribution (mm³) of CPN and BPN genevectors. * Denotes statistical significance P<0.05.

FIGS. 6A and 6B are a box graph (FIG. 6A) and a histogram (FIG. 6B),respectively, showing image based Matlab quantification of volume ofdistribution of eGFP expression (mm³) for each of UPN, CPN and BPN genevectors. (N=4-6). Data represents the mean±SEM. *Denotes statisticalsignificance P<0.05.

FIG. 7 is a histogram showing normalized GFP expression by UPN, CPN andBPN nanoparticle gene vectors in rodent striatum following CEDadministration. The expression level of GFP was normalized with β-actin.Data represents the mean±SEM. * Denotes statistical significance P<0.05.

FIG. 8 is a histogram showing volume of distribution (mm³) for CPN andBPN following CED at a concentration of 0.5 mg/ml and 1 mg/ml,respectively. *p<0.05.

FIG. 9 is a schematic showing the click chemistry reaction for synthesisof PEGylated PLL polymers containing branched and unbranched PEG.

FIG. 10 is a graph showing the aspect ratio (major diameter/minordiameter) of nanoparticles formed from Poly L-lysine (PLL), PEG, ordifferent percentages of Poly L-lysine and branched PEG (BR 50, BR 90 orBR 100) with nitrogen (polymer) to phosphorous (nucleic acid) (N/P)ratios of 2 or 5, respectively. * Denotes statistical significance indifferences with PLL-PEG gene vectors, p value<0.05.

FIGS. 11A to 11D are a histograms showing the percentage of particles(%) over size (nm) of the major diameter of particles consisting of PolyL-lysine and PEG (PLL-PEG) (FIG. 11A), Poly L-lysine and 100% branchedPEG (BR 100) (FIG. 11B), Poly L-lysine and 50% branched PEG (BR 50)(FIG. 11C) and Poly L-lysine and 90% branched PEG (BR 90) (FIG. 11D),respectively. The vertical bar represents a cut-off of 114 nm.

FIG. 12 is a graph showing the % cell viability over concentration ofplasmid (μg/ml) for PLL N/P 2 (

), PLL N/P 5 (

), PLL-PEG N/P 2 (

), PLL-PEG N/P 5 (

), BR 90 N/P 2 (

) and BR 90 N/P (

), respectively. Error bars depict SEM.

FIGS. 13A and 13B are histograms showing the percentage of cellviability (%) for PLL-PEG, BR 100 and BR 90 at concentrations of 1μg/ml, 5 μg/ml and 10 μg/ml, for 9 L glioma cells (FIG. 13 A), andprimary rat astrocyte cells (FIG. 13 B), respectively. Error bars depictSEM.

FIGS. 14A to 14D are histograms showing the percentage of cellularuptake (%) for pBal PLL-PEG, PLL BR 100 and BR 90, for 9 L glioma cells(FIG. 14A), and primary rat astrocyte cells (FIG. 14B), and luciferasegene expression (RLU/mg of protein) following in vitro gene vectortransfection of 9 L glioma cells (FIG. 14C), and primary rat astrocytecells (FIG. 14D), respectively. Data represents the mean±SEM. * Denotesstatistical significance P<0.05.

FIGS. 15A and 15B are histograms showing the gene vector Z-average (nm)(FIG. 15A) and polydispersity (PDI) (FIG. 15B), respectively, forPLL-PEG, BR 100 and BR 90 at pretreatment, 0 hour and one hour aftertreatment with aCSF at 37° C.

FIG. 16 is a graph showing the ensemble-averaged geometric mean squaredisplacements <MSD> (μm²) over timescale (Sec) for PLL, PLL-PEG, BR 100and BR 90, respectively. Data represent the ensemble average of at leastthree independent experiments with N≥500 particles tracked for eachexperiment, error bars depict SEM.

FIG. 17 is a schematic showing the representative particle trajectoriesover 20 s. Trajectories shown are of particles that had an MSD equal tothe ensemble average at a time scale of 1, Scale bar=0.25 μm.

FIG. 18 is a panel of histograms showing Percentage Gene Vector (%) overlog <MSD> for gene vectors for PLL, PLL-PEG, BR 100 and BR 90,respectively. Data are taken from at least three independent experimentsat a timescale of 1 s.

FIG. 19 is a graph showing area of distribution (mm²) of gene vectors asa function of distance from injection site (mm) for BR90 (●) and PLL-PEG(∘) respectively, quantified from individual confocal images of 100 μmslices of brain tissue. N=3, error bars represent standard error inmean.

FIG. 20 is a histogram showing volume of distribution of gene vectors(mm³) for BR90 and PLL-PEG, respectively, quantified from confocalimages of 100 μm slices of brain tissue. * Denotes statisticalsignificance, P value<0.05.

FIG. 21 is a bar graph demonstrating the volume of distribution (mm³) ofPEI CPN and BPN at plasmid concentrations of 0.5 mg/mL and 1 mg/mL. Dataare taken from six independent experiments. * Denotes statisticalsignificance of p<0.05.

FIG. 22A is a bar graph showing fluorescence intensity per brain sliceat different distances (μm) from injection plane following injection ofBR90 (DNA-BNP) or PLL-PEG (DNA-CNP). FIG. 22B is a bar graph showing thenumber of cells transfected per brain slice at different distances (μm)from injection plane following injection of BR90 (DNA-BNP) or PLL-PEG(DNA-CNP). * Denotes statistical significance p<0.05.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms “biocompatible” as used herein refers to one or more materialsthat are neither themselves toxic to the host (e.g., an animal orhuman), nor degrade (if the polymer degrades) at a rate that producesmonomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host.

The term “biodegradable” as used herein means that the materialsdegrades or breaks down into its component subunits, or digestion, e.g.,by a biochemical process, of the polymer into smaller, non-polymericsubunits.

The term “corresponding particle” or “reference particles” as usedherein refers to a particle that is substantially identical to anotherparticle to which it is compared, but typically lacking a surfacemodification to promote transport differences through the pores in theECM of the brain. A corresponding particle is typically of similarmaterial, density, and size as the particle to which it is compared. Incertain embodiments, a corresponding particle is a particle that doesnot have a dense coating of polyethylene glycol. In certain embodiments,a comparable particle is a particle that is not formed of a blendedmixture containing free polymer and polymer conjugated to polyethyleneglycol.

The term “densely coated particle” refers to a particle that is modifiedto specifically enhance the density of coating agent at the surface ofthe particle, for example, relative to a reference particle. In someembodiments, a densely coated particle is formed from a ratio ofpolyethylene glycol to polymer that is sufficient to alter thephysicochemical properties of the particle relative to a less denselycoated, or non-coated particle. In some embodiments, the density ofcoating agent is sufficient to completely mask the charge of theparticle, resulting in a near neutral charge and near neutral zetapotential value and colloidal stability in physiological solutions. In aparticular embodiment, a densely coated particle is achieved usingbranched polyethylene glycol or branched polymer, wherein the branchingenhances the ratio of polyethylene glycol to polymer as compared to areference particle that does not contain a branched polymer or branchedpolyethylene glycol.

The term “nucleic acids” refers to isolated DNA, cDNA, RNA, miRNA,siRNA, plasmids, vectors, and expression constructs.

The term “diameter” is art-recognized and is used herein to refer toeither of the physical diameter or the hydrodynamic diameter. Thediameter of an essentially spherical particle may refer to the physicalor hydrodynamic diameter. The diameter of a non-spherical particle mayrefer preferentially to the hydrodynamic diameter. As used herein, thediameter of a non-spherical particle may refer to the largest lineardistance between two points on the surface of the particle. Whenreferring to multiple particles, the diameter of the particles typicallyrefers to the average diameter of the particles.

“Sustained release” as used herein refers to release of a substance overan extended period of time in contrast to a bolus type administration inwhich the entire amount of the substance is made biologically availableat one time.

The term “microspheres”, “microparticles”, and “microcapsules” are usedinterchangeably unless otherwise stated. These have a size between aboutone up to about 1000 microns. In general, “microcapsules,” have a coreof a different material than the shell material. A microparticle may bespherical or nonspherical and may have any regular or irregular shape.If the structures are less than about one micron in diameter, then thecorresponding art-recognized terms “nanosphere,” “nanocapsule,” and“nanoparticle” may be utilized. In certain embodiments, the nanospheres,nanocapsules and nanoparticles have an average diameter of about 100 nm,or less than 100 nm, such as 50 nm, or 10 nm.

A composition comprising microparticles or nanoparticles may includeparticles of a range of particle sizes. In certain embodiments, theparticle size distribution may be uniform, e.g., within less than abouta 20% standard deviation of the median volume diameter, and in otherembodiments, still more uniform, e.g., within about 10% of the medianvolume diameter.

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and includewithout limitation intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The term “surfactant” refers to an agent that lowers the surface tensionof a liquid.

The term “therapeutic agent” refers to an agent that can be administeredto prevent or treat a disease or disorder. Examples include, but are notlimited to, a nucleic acid, a nucleic acid analog, a small molecule, apeptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, orsurfactant, or a combination thereof.

The term “treating” refers to preventing or alleviating one or moresymptoms of a disease, disorder or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected, such as treating the pain of a subject by administration of ananalgesic agent even though such agent does not treat the cause of thepain.

The phrase “pharmaceutically acceptable” refers to compositions,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” refers to pharmaceuticallyacceptable materials, compositions or vehicles, such as a liquid orsolid filler, diluent, solvent or encapsulating material involved incarrying or transporting any subject composition, from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of a subject composition and not injurious to thepatient.

The phrase “therapeutically effective amount” refers to an amount of thetherapeutic agent that produces some desired effect at a reasonablebenefit/risk ratio applicable to any medical treatment. The effectiveamount may vary depending on such factors as the disease or conditionbeing treated, the particular targeted constructs being administered,the size of the subject, or the severity of the disease or condition.One of ordinary skill in the art may empirically determine the effectiveamount of a particular compound without necessitating undueexperimentation.

The terms “incorporated” and “encapsulated” refer to incorporating,formulating, or otherwise including an active agent into and/or onto acomposition that allows for release, such as sustained release, of suchagent in the desired application. The terms contemplate any manner bywhich a therapeutic agent or other material is incorporated into apolymer matrix, including chemically or physically couple, in physicaladmixture, or enveloping the agent in a coating layer

II. Compositions

Synthetic gene delivery platforms with a dense surface coating ofhydrophilic and neutrally charged polymer such as polyethylene glycol(PEG) or polyethylene glycol-polyoxyethylene block copolymer known aspoloxamer such as a PLURONIC® (referred to collectively as “PEGylatedgene vectors”) which are capable of rapid diffusion and widespreaddistribution in brain tissue are disclosed.

A. Nanoparticles

In some embodiments the densely PEGylated gene vectors are nanoparticlesformulated from a mixture of unconjugated and polyethylene glycol (PEG)conjugated cationic polymers. The nanoparticles rapidly penetrate thebrain parenchyma and have reduced cytotoxicity and increased colloidalstability in high ionic strength solutions, relative to less-denselyPEGylated gene vectors.

1. Coating Agents

Nanoparticles, coated with one or more materials that promote diffusionof the particles through the ECM in the brain by reducing interactionsbetween the particles and brain tissue (e.g., surface altering agents),are disclosed. Examples of the surface-altering agents include, but arenot limited to, polyethylene glycol (“PEG”) and poloxomers (polyethyleneoxide block copolymers).

i. Polyethylene Glycol (PEG)

A preferred coating agent is poly(ethylene glycol), also known as PEG.PEG may be employed to reduce adhesion in brain ECM in certainconfigurations, e.g., wherein the length of PEG chains extending fromthe surface is controlled (such that long, unbranched chains thatinterpenetrate into the ECM are reduced or eliminated). For example,linear high MW PEG may be employed in the preparation of particles suchthat only portions of the linear strands extend from the surface of theparticles (e.g., portions equivalent in length to lower MW PEGmolecules). Alternatively, branched high MW PEG may be employed. In suchembodiments, although the molecular weight of a PEG molecule may behigh, the linear length of any individual strand of the molecule thatextends from the surface of a particle would correspond to a linearchain of a lower MW PEG molecule.

Representative PEG molecular weights in daltons (Da) include 300 Da, 600Da, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 8 kDa, 10 kDa, 15 kDa, 20kDa, 30 kDa, 50 kDa, 100 kDa, 200 kDa, 500 kDa, and 1 MDa. In preferredembodiments, the PEG has a molecular weight of about 5,000 Daltons. PEGof any given molecular weight may vary in other characteristics such aslength, density, and branching. In a particular embodiment, a coatingagent is methoxy-PEG-amine, with a MW of 5 kDa. In another embodiment, acoating agent is methoxy-PEG-N-hydroxysuccinimide with a MW of 5 kDa(mPEG-NHS 5 kDa).

In alternative embodiments, the coating is a poloxamer such as thepolyethylene glycol-polyethylene oxide block copolymers marketed asPLUORONICs®.

iii. Density of Coating Agent

In preferred embodiments the nanoparticles are coated with PEG or othercoating agent at a density that optimizes rapid diffusion through thebrain parenchyma. The density of the coating can be varied based on avariety of factors including the material and the composition of theparticle.

In a preferred embodiment the co-polymer molar ratio of PEG or othercoating agent to cationic polymer is greater than 8 (i.e., more than 8moles of PEG to every mole of cationic polymer). The ratio by moles ofPEG or other coating agent to cationic polymer can be 9, 10, 11, 12, 13,14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 37,50 or more than 50. A preferred molar ratio of PEG or other coatingagent to cationic polymer is 26.

In one embodiment, the density of the PEG or other coating agent is atleast 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10,20, 50, or 100 units per nm².

In another embodiment, the amount of the PEG or other coating agent isexpressed as a percentage of the mass of the particle. In a particularembodiment, the mass of the PEG or other coating agent is at least1/10,000, 1/7500, 1/5000, 1/4000, 1/3400, 1/2500, 1/2000, 1/1500,1/1000, 1/500, 1/250, 1/200, 1/150, 1/100, 1/75, 1/50, 1/25, 1/20, ⅕, ½,or 9/10 of the mass of the particle. In a further embodiment, the weightpercent of the PEG or other coating agent is at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, orgreater.

2. Core Polymer

Any number of biocompatible polymers can be used to prepare thenanoparticles. In preferred embodiments, the biocompatible polymer(s) isa cationic polymer. The cationic polymer can be a branched polymer, toenhance the capacity of the polymer to conjugate to a coating agent suchas PEG. In some embodiments, the biocompatible polymer(s) isbiodegradable.

i. Types of Polymers

Exemplary cationic polymers include, but are not limited to,cyclodextrin-containing polymers, in particular cationiccyclodextrin-containing polymers, such as those described in U.S. Pat.No. 6,509,323, polyethylenimine (PEI), poly(L-lysine) (PLL),polyethylenimine (PEI), polymethacrylate, chitosan,poly(glycoamidoamine), schizophyllan, DEAE-dextran, dextran-spermine,poly(amido-amine) (PAA), poly(4-hydroxy-L-proline ester),poly[R-(4-aminobutyl)-L-glycolic acid] (PAGA), poly(amino-ester),poly(phosphazenes) (PPZ), poly(phosphoesters) (PPE),poly(phosphoramidates) (PPA), TAT-based peptides, Antennapediahomeodomain peptide, MPG peptide, poly(propylenimine), carbosilane, andamine-terminated polyaminophosphine. In a particular embodiment thepolymer is a cationic polymer with multiple free amines. Preferredpolymers include polyethylenimine (PEI) and poly-L-lysine (PLL).Copolymers of two or more polymers described above, including blockand/or random copolymers, may also be employed to make the polymericparticles.

ii. Branched Polymers

In polymer chemistry, branching occurs by the replacement of asubstituent, e.g., a hydrogen atom, on a monomer subunit, by anothercovalently bonded chain of that polymer; or, in the case of a graftcopolymer, by a chain of another type. Branching may result from theformation of carbon-carbon or various other types of covalent bonds.Branching by ester and amide bonds is typically by a condensationreaction, producing one molecule of water (or HCl) for each bond formed.

The branching index measures the effect of long-chain branches on thesize of a macromolecule in solution. It is defined as g=<sb2>/<sl2>,where sb is the mean square radius of gyration of the branchedmacromolecule in a given solvent, and sl is the mean square radius ofgyration of an otherwise identical linear macromolecule in the samesolvent at the same temperature. A value greater than 1 indicates anincreased radius of gyration due to branching.

In preferred embodiments, the core polymer or PEG is a branched polymerthat is capable of enhancing conjugation of the coating agent and corepolymer. Exemplary branched polymers include 25 kDa branchedpolyethyleneimine (PEI) and 5 kDa branched methoxy-PEG.

iii. Copolymers

In preferred embodiments, copolymers of PEG or derivatives thereof withany of the polymers described above may be used to make the polymericparticles. In certain embodiments, the PEG or derivatives may locate inthe interior positions of the copolymer. Alternatively, the PEG orderivatives may locate near or at the terminal positions of thecopolymer. In certain embodiments, the nanoparticles are formed underconditions that allow regions of PEG to phase separate or otherwiselocate to the surface of the particles. The surface-localized PEGregions alone may perform the function of, or include, asurface-altering agent.

3. Nucleic Acids

Nanoparticle gene carriers typically carry one or more nucleic acids.The nucleic acid can alter, correct, or replace an endogenous nucleicacid sequence. In preferred embodiments, the nucleic acid is used totreat cancers, correct defects in genes in brain diseases and metabolicdiseases affecting brain function, genes such as those for the treatmentof Parkinsons and ALS.

Gene therapy is a technique for correcting defective genes responsiblefor disease development. Researchers may use one of several approachesfor correcting faulty genes:

-   -   A normal gene may be inserted into a nonspecific location within        the genome to replace a nonfunctional gene. This approach is        most common.    -   An abnormal gene could be swapped for a normal gene through        homologous recombination.    -   The abnormal gene could be repaired through selective reverse        mutation, which returns the gene to its normal function.    -   The regulation (the degree to which a gene is turned on or off)        of a particular gene could be altered.

The nucleic acid carried by the nanoparticle gene carrier can be a DNA,RNA, a chemically modified nucleic acid, or combinations thereof. Forexample, methods for increasing stability of nucleic acid half-life andresistance to enzymatic cleavage are known in the art, and can includeone or more modifications or substitutions to the nucleobases, sugars,or linkages of the polynucleotide. For example, the nucleic acid can becustom synthesized to contain properties that are tailored to fit adesired use. Common modifications include, but are not limited to, useof locked nucleic acids (LNAs), unlocked nucleic acids (UNAs),morpholinos, peptide nucleic acids (PNA), phosphorothioate linkages,phosphonoacetate linkages, propyne analogs, 2′-O-methyl RNA, 5-Me-dC,2′-5′ linked phosphodiester linage, Chimeric Linkages (Mixedphosphorothioate and phosphodiester linkages and modifications),conjugation with lipid and peptides, and combinations thereof.

In some embodiments, the nucleic acid includes intemucleotide linkagemodifications such as phosphate analogs having achiral and unchargedintersubunit linkages (e.g., Sterchak, E. P. et al., Organic Chem.,52:4202, (1987)), or uncharged morpholino-based polymers having achiralintersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506). Someinternucleotide linkage analogs include morpholidate, acetal, andpolyamide-linked heterocycles. Other backbone and linkage modificationsinclude, but are not limited to, phosphorothioates, peptide nucleicacids, tricyclo-DNA, decoy oligonucleotide, ribozymes, spiegelmers(containing L nucleic acids, an apatamer with high binding affinity), orCpG oligomers.

Phosphorothioates (or S-oligos) are a variant of normal DNA in which oneof the non-bridging oxygens is replaced by sulfur. The sulfurization ofthe internucleotide bond dramatically reduces the action of endo- andexonucleases including 5′ to 3′ and 3′ to 5′ DNA POL 1 exonuclease,nucleases S1 and P1, RNases, serum nucleases and snake venomphosphodiesterase. In addition, the potential for crossing the lipidbilayer increases. Because of these important improvements,phosphorothioates have found increasing application in cell regulation.Phosphorothioates are made by two principal routes: by the action of asolution of elemental sulfur in carbon disulfide on a hydrogenphosphonate, or by the more recent method of sulfurizing phosphitetriesters with either tetraethylthiuram disulfide (TETD) or3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD). The latter methods avoidthe problem of elemental sulfur's insolubility in most organic solventsand the toxicity of carbon disulfide. The TETD and BDTD methods alsoyield higher purity phosphorothioates. (See generally Uhlmann andPeymann, 1990, Chemical Reviews 90, at pages 545-561 and referencescited therein, Padmapriya and Agrawal, 1993, Bioorg. & Med. Chem. Lett.3, 761).

Peptide nucleic acids (PNA) are molecules in which the phosphatebackbone of oligonucleotides is replaced in its entirety by repeatingN-(2-aminoethyl)-glycine units and phosphodiester bonds are replaced bypeptide bonds. The various heterocyclic bases are linked to the backboneby methylene carbonyl bonds. PNAs maintain spacing of heterocyclic basesthat is similar to oligonucleotides, but are achiral and neutrallycharged molecules. Peptide nucleic acids are typically comprised ofpeptide nucleic acid monomers. The heterocyclic bases can be any of thestandard bases (uracil, thymine, cytosine, adenine and guanine) or anyof the modified heterocyclic bases described below. A PNA can also haveone or more peptide or amino acid variations and modifications. Thus,the backbone constituents of PNAs may be peptide linkages, oralternatively, they may be non-peptide linkages. Examples include acetylcaps, and amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referredto herein as O-linkers). Methods for the chemical assembly of PNAs arewell known. See, for example, U.S. Pat. Nos. 5,539,082, 5,527,675,5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.

In some embodiments, the nucleic acid includes one or morechemically-modified heterocyclic bases including, but not limited to,inosine, 5-(1-propynyl) uracil (pU), 5-(1-propynyl) cytosine (pC),5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5and 2-amino-5-(2′-deoxy-β-D-ribofuranosyl)pyridine (2-aminopyridine),and various pyrrolo- and pyrazolopyrimidine derivatives,4-acetylcytosine, 8-hydroxy-N-6-methyladeno sine, aziridinylcytosine,5-(carboxyhydroxylmethyl) uracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methyl guanine,1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,3-methylcytosine, N6-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil,5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyaceticacid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,2,6-diaminopurine, and 2′-modified analogs such as, but not limited to,O-methyl, amino-, and fluoro-modified analogs Inhibitory RNAs modifiedwith 2′-flouro (2′-F) pyrimidines appear to have favorable properties invitro. Moreover, one report suggested 2′-F modified siRNAs have enhancedactivity in cell culture as compared to 2′-OH containing siRNAs. 2′-Fmodified siRNAs are functional in mice but that they do not necessarilyhave enhanced intracellular activity over 2′-OH siRNAs.

In some embodiments the nucleic acid includes one or more sugar moietymodifications, including, but not limited to, 2′-O-aminoethoxy,2′-O-amonioethyl (2′-OAE), 2′-O-methoxy, 2′-O-methyl, 2-guanidoethyl(2′-OGE), 2′-0,4′-C-methylene (LNA), 2′-O-(methoxyethyl) (2′-OME) and2′-O—(N-(methyl)acetamido) (2′-OMA).

Nanoparticle gene carriers carrying one or more nucleic acid can beutilized to deliver nucleic acid cargo in a method of gene therapy.Methods of gene therapy typically rely on the introduction into the cellof a nucleic acid molecule that alters the genotype of the cell. Forexample, corrective genes can be introduced into a non-specific locationwithin the host's genome. This approach typically requires deliverysystems to introduce the replacement gene into the cell, such asgenetically engineered viral vectors.

In other embodiments, functional nucleic acids are introduced to preventthe function or expression of a particular gene that causes a defect ordisease.

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. For example, functional nucleic acids include, but are notlimited to, antisense molecules, siRNA, miRNA, aptamers, ribozymes,triplex forming molecules, RNAi, and external guide sequences. Thefunctional nucleic acid molecules can act as effectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA or the genomic DNA of a targetpolypeptide or they can interact with the polypeptide itself. Oftenfunctional nucleic acids are designed to interact with other nucleicacids based on sequence homology between the target molecule and thefunctional nucleic acid molecule. In other situations, the specificrecognition between the functional nucleic acid molecule and the targetmolecule is not based on sequence homology between the functionalnucleic acid molecule and the target molecule, but rather is based onthe formation of tertiary structure that allows specific recognition totake place.

In a particular embodiment, the inhibitory nucleic acids are antisensenucleic acids. Antisense molecules are designed to interact with atarget nucleic acid molecule through either canonical or non-canonicalbase pairing. The interaction of the antisense molecule and the targetmolecule promotes the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule interrupts a processing function that normally wouldtake place on the target molecule, such as transcription or replication.Antisense molecules can be designed based on the sequence of the targetmolecule. Numerous methods for optimization of antisense efficiency byfinding the most accessible regions of the target molecule exist.Exemplary methods would be in vitro selection experiments and DNAmodification studies using DMS and DEPC. It is preferred that antisensemolecules bind the target molecule with a dissociation constant (K_(d))less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹².

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP and theophiline, as well as large molecules, suchas reverse transcriptase and thrombin. Aptamers can bind very tightlywith K_(d)'s from the target molecule of less than 10-12 M. It ispreferred that the aptamers bind the target molecule with a K_(d) lessthan 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². Aptamers can bind the target moleculewith a very high degree of specificity. For example, aptamers have beenisolated that have greater than a 10,000 fold difference in bindingaffinities between the target molecule and another molecule that differat only a single position on the molecule. It is preferred that theaptamer have a K_(d) with the target molecule at least 10, 100, 1000,10,000, or 100,000 fold lower than the K_(d) with a background bindingmolecule. It is preferred when doing the comparison for a polypeptidefor example, that the background molecule be a different polypeptide.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly. It ispreferred that the ribozymes catalyze intermolecular reactions. Thereare a number of different types of ribozymes that catalyze nuclease ornucleic acid polymerase type reactions which are based on ribozymesfound in natural systems, such as hammerhead ribozymes. There are also anumber of ribozymes that are not found in natural systems, but whichhave been engineered to catalyze specific reactions de novo. Preferredribozymes cleave RNA or DNA substrates, and more preferably cleave RNAsubstrates. Ribozymes typically cleave nucleic acid substrates throughrecognition and binding of the target substrate with subsequentcleavage. This recognition is often based mostly on canonical ornon-canonical base pair interactions. This property makes ribozymesparticularly good candidates for target specific cleavage of nucleicacids because recognition of the target substrate is based on the targetsubstrates sequence. Triplex forming functional nucleic acid moleculesare molecules that can interact with either double-stranded orsingle-stranded nucleic acid. When triplex molecules interact with atarget region, a structure called a triplex is formed, in which thereare three strands of DNA forming a complex dependent on bothWatson-Crick and Hoogsteen base-pairing. Triplex molecules are preferredbecause they can bind target regions with high affinity and specificity.It is preferred that the triplex forming molecules bind the targetmolecule with a K_(d) less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹².

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. Similarly,eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized tocleave desired targets within eukarotic cells. Representative examplesof how to make and use EGS molecules to facilitate cleavage of a varietyof different target molecules are known in the art.

Gene expression can also be effectively silenced in a highly specificmanner through RNA interference (RNAi). This silencing was originallyobserved with the addition of double stranded RNA (dsRNA) (Fire, A., etal. (1998) Nature, 391:806-11; Napoli, C., et al. (1990) Plant Cell2:279-89; Hannon, G. J. (2002) Nature, 418:244-51). Once dsRNA enters acell, it is cleaved by an RNase III-like enzyme, Dicer, into doublestranded small interfering RNAs (siRNA) 21-23 nucleotides in length thatcontains 2 nucleotide overhangs on the 3′ ends (Elbashir, S. M., et al.(2001) Genes Dev., 15:188-200; Bernstein, E., et al. (2001) Nature,409:363-6; Hammond, S. M., et al. (2000) Nature, 404:293-6). In an ATPdependent step, the siRNAs become integrated into a multi-subunitprotein complex, commonly known as the RNAi induced silencing complex(RISC), which guides the siRNAs to the target RNA sequence (Nykanen, A.,et al. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds,and it appears that the antisense strand remains bound to RISC anddirects degradation of the complementary mRNA sequence by a combinationof endo and exonucleases (Martinez, J., et al. (2002) Cell, 110:563-74).However, the effect of iRNA or siRNA or their use is not limited to anytype of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can inducesequence-specific post-transcriptional gene silencing, therebydecreasing or even inhibiting gene expression. In one example, an siRNAtriggers the specific degradation of homologous RNA molecules, such asmRNAs, within the region of sequence identity between both the siRNA andthe target RNA. For example, WO 02/44321 discloses siRNAs capable ofsequence-specific degradation of target mRNAs when base-paired with 3′overhanging ends, herein incorporated by reference for the method ofmaking these siRNAs. Sequence specific gene silencing can be achieved inmammalian cells using synthetic, short double-stranded RNAs that mimicthe siRNAs produced by the enzyme dicer (Elbashir, S. M., et al. (2001)Nature, 411:494 498) (Ui-Tei, K., et al. (2000) FEBS Lett. 479:79-82).siRNA can be chemically or in vitro-synthesized or can be the result ofshort double-stranded hairpin-like RNAs (shRNAs) that are processed intosiRNAs inside the cell. Synthetic siRNAs are generally designed usingalgorithms and a conventional DNA/RNA synthesizer. Suppliers includeAmbion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette,Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg,Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands).siRNA can also be synthesized in vitro using kits such as Ambion'sSILENCER® siRNA Construction Kit.

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAs (shRNAs). Kits for the productionof vectors comprising shRNA are available, such as, for example,Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™inducible RNAi plasmid and lentivirus vectors.

An miRNA or pre-miRNA can be 18-100 nucleotides in length, and morepreferably from 18-80 nucleotides in length. Mature miRNAs can have alength of 19-30 nucleotides, preferably 21-25 nucleotides, particularly21, 22, 23, 24, or 25 nucleotides. MicroRNA precursors typically have alength of about 70-100 nucleotides and have a hairpin conformation.

Given the sequence of an miRNA or a pre-miRNA, an miRNA antagonist thatis sufficiently complementary to a portion of the miRNA or a pre-miRNAcan be designed according to the rules of Watson and Crick base pairing.As used herein, the term “sufficiently complementary” means that twosequences are sufficiently complementary such that a duplex can beformed between them under physiologic conditions. An miRNA antagonistsequence that is sufficiently complementary to an miRNA or pre-miRNAtarget sequence can be 70%, 80%, 90%, or more identical to the miRNA orpre-miRNA sequence. In one embodiment, the miRNA antagonist contains nomore than 1, 2 or 3 nucleotides that are not complementary to the miRNAor pre-miRNA target sequence. In a preferred embodiment, the miRNAantagonist is 100% complementary to an miRNA or pre-miRNA targetsequence. In some embodiments, the miRNA antagonist is complementary toa portion of the miRNA or pre-miRNA sequence of a human. Sequences formiRNAs are available publicly, for example, through the miRBase registry(Griffiths-Jones, et al., Nucleic Acids Res., 36(DatabaseIssue):D154-D158 (2008); Griffiths-Jones, et al., Nucleic Acids Res.,36(Database Issue):D140-D144 (2008); Griffiths-Jones, et al., NucleicAcids Res., 36(Database Issue):D109-D111 (2008)) and other publicallyaccessible databases.

In some embodiments, there will be nucleotide mismatches in the regionof complementarity. In a preferred embodiment, the region ofcomplementarity will have no more than 1, 2, 3, 4, or 5 mismatches.

In one embodiment, the miRNA antagonists are oligomers or polymers ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or modificationsthereof. miRNA antagonists include oligonucleotides that containnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages.

In some embodiments, the miRNA antagonists are antagomirs. Antagomirsare a specific class of miRNA antagonists that are described, forexample, in US2007/0213292 to Stoffel et al. Antagomirs are RNA-likeoligonucleotides that contain various modifications for RNase protectionand pharmacologic properties such as enhanced tissue and cellularuptake. Antagomirs differ from normal RNA by having complete2′-O-methylation of sugar, phosphorothioate backbone and acholesterol-moiety at 3′-end.

Antagomirs can include a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. In one embodiment, antagomirs contain six phosphorothioatebackbone modifications; two phosphorothioates are located at the 5′-endand four at the 3′-end. Phosphorothioate modifications provideprotection against RNase activity and their lipophilicity contributes toenhanced tissue uptake.

Examples of antagomirs and other miRNA inhibitors are described inWO2009/020771, WO2008/091703, WO2008/046911, WO2008/074328,WO2007/090073, WO2007/027775, WO2007/027894, WO2007/021896,WO2006/093526, WO2006/112872, WO2007/112753, WO2007/112754,WO2005/023986, or WO2005/013901, all of which are hereby incorporated byreference.

Custom designed Anti-miR™ molecules are commercially available fromApplied Biosystems. Thus, in some embodiments, the antagomir is anAmbion® Anti-miR™ inhibitor. These molecules are chemically modified andoptimized single-stranded nucleic acids designed to specifically inhibitnaturally occurring mature miRNA molecules in cells.

Custom designed Dharmacon Meridian™ microRNA Hairpin Inhibitors are alsocommercially available from Thermo Scientific. These inhibitors includechemical modifications and secondary structure motifs. For example,Vermeulen et al. reports in US2006/0223777 the identification ofsecondary structural elements that enhance the potency of thesemolecules. Specifically, incorporation of highly structured,double-stranded flanking regions around the reverse complement coresignificantly increases inhibitor function and allows for multi-miRNAinhibition at subnanomolar concentrations. Other such improvements inantagomir design are contemplated for use in the disclosed methods.

Methods to construct expression vectors containing genetic sequences andappropriate transcriptional and translational control elements are wellknown in the art. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Expression vectors generally contain regulatory sequences necessaryelements for the translation and/or transcription of the inserted codingsequence. For example, the coding sequence is preferably operably linkedto a promoter and/or enhancer to help control the expression of thedesired gene product. Promoters used in biotechnology are of differenttypes according to the intended type of control of gene expression. Theycan be generally divided into constitutive promoters, tissue-specific ordevelopment-stage-specific promoters, inducible promoters, and syntheticpromoters.

Gene targeting via target recombination, such as homologousrecombination (HR), is another strategy for gene correction. Genecorrection at a target locus can be mediated by donor DNA fragmentshomologous to the target gene (Hu, et al., Mol. Biotech., 29:197-210(2005); Olsen, et al., J. Gene Med., 7:1534-1544 (2005)). One method oftargeted recombination includes the use of triplex-formingoligonucleotides (TFOs) which bind as third strands tohomopurine/homopyrimidine sites in duplex DNA in a sequence-specificmanner. Triplex forming oigonucleotides can interact with eitherdouble-stranded or single-stranded nucleic acids. When triplex moleculesinteract with a target region, a structure called a triplex is formed,in which there are three strands of DNA forming a complex dependent onboth Watson-Crick and Hoogsteen base-pairing. Triplex molecules arepreferred because they can bind target regions with high affinity andspecificity. It is preferred that the triplex forming molecules bind thetarget molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12.

Methods for targeted gene therapy using triplex-forming oligonucleotides(TFO's) and peptide nucleic acids (PNAs) are described in U.S. PublishedApplication No. 20070219122 and their use for treating infectiousdiseases such as HIV are described in U.S. Published Application No.2008050920. The triplex-forming molecules can also be tail clamp peptidenucleic acids (tcPNAs), such as those described in U.S. PublishedApplication No. 2011/0262406. Highly stable PNA:DNA:PNA triplexstructures can be formed from strand invasion of a duplex DNA with twoPNA strands. In this complex, the PNA/DNA/PNA triple helix portion andthe PNA/DNA duplex portion both produce displacement of thepyrimidine-rich triple helix, creating an altered structure that hasbeen shown to strongly provoke the nucleotide excision repair pathwayand to activate the site for recombination with the donoroligonucleotide. Two PNA strands can also be linked together to form abis-PNA molecule. The triplex-forming molecules are useful to inducesite-specific homologous recombination in mammalian cells when used incombination with one or more donor oligonucleotides which provides thecorrected sequence. Donor oligonucleotides can be tethered totriplex-forming molecules or can be separate from the triplex-formingmolecules. The donor oligonucleotides can contain at least onenucleotide mutation, insertion or deletion relative to the target duplexDNA.

Double duplex-forming molecules, such as a pair of pseudocomplementaryoligonucleotides, can also induce recombination with a donoroligonucleotide at a chromosomal site. Use of pseudocomplementaryoligonucleotides in targeted gene therapy is described in U.S. PublishedApplication No. 2011/0262406. Pseudocomplementary oligonucleotides arecomplementary oligonucleotides that contain one or more modificationssuch that they do not recognize or hybridize to each other, for exampledue to steric hindrance, but each can recognize and hybridize tocomplementary nucleic acid strands at the target site. In someembodiments, pseudocomplementary oligonucleotides are pseudocomplemenarypeptide nucleic acids (pcPNAs). Pseudocomplementary oligonucleotides canbe more efficient and provide increased flexibility over methods ofinduced recombination such as triple-helix oligonucleotides andbis-peptide nucleic acids which require a polypurine sequence in thetarget double-stranded DNA.

The molar ratio of the nucleic acid to the core polymer within thenanoparticles can be at least 0.5, 1, 10, 100, 1000 or more than 1000.

4. Additional Active Agents

Nanoparticle gene carriers may carry only “genetic” materials, or othertherapeutic, prophylactic and/or diagnostic agents can be co-delivereddepending on the application. However, any “genetic” materials that canperform the listed functions can be packaged into the nanoparticles. Forexample, tumor suppressor genes such as p53 and Rb can be complexed intonanoparticles to be used for cancer patients, so as any plasmid DNA orsiRNA that possess anti-inflammatory, anti-viral functions, etc.

These additional; active agents can be dispersed in the nanoparticlegene carriers or be covalently attached to one or more of the polymericcomponents of the nanoparticle.

Suitable additional active agents include, but are not limited to, othernucleic acid-based medicine, anti-inflammatory drugs,antiproliferatives, chemotherapeutics, vasodilators, and anti-infectiveagents. In certain embodiments, the nanoparticle gene carriers containone or more antibiotics, such as tobramycin, colistin, or aztreonam. Thedisclosed nanoparticle gene carriers can optionally contain one or moreantibiotics which are known to possess anti-inflammatory activity, suchas erythromycin, azithromycin, or clarithromycin. Nanoparticles may alsobe used for the delivery of chemotherapeutic agents, andanti-proliferative agents.

5. Nanoparticle Properties

As shown in the examples, the disclosed nanoparticles diffuse throughthe pores of the ECM of the brain at a greater rate of diffusivity thana reference nanoparticle, such as an uncoated particle, e.g., uncoatedPEI particle.

i. Particle Diffusivity

The disclosed nanoparticles may pass through the pores of the ECM of thebrain at a rate of diffusivity that is at least 5, 10, 20, 30, 50, 60,80, 100, 125, 150, 200, 250, 500, 600, 750, 1000, 1500, 2000, 2500,3000, 4000, 5000, 10000- or greater fold higher than a referenceparticle.

The transport rates of the particles can be measured using a variety oftechniques in the art. In one embodiment, the rate of diffusion ismeasured by geometric ensemble mean squared displacements (MSD). In aparticular embodiment, the particles may diffuse through the pores ofthe ECM of the brain with an MSD that is at least 5, 10, 20, 30, 50, 60,80, 100, 125, 150, 200, 250, 500, 600, 750, 1000, 1500, 2000, 2500,3000, 4000, 5000, 10000- or greater fold higher than a referenceparticle.

In other embodiments, the disclosed nanoparticles diffuse through thepores of the ECM of the brain at a rate approaching the rate ofdiffusivity at which the particles diffuse through water. In aparticular embodiment, the rate of diffusivity is at least 1/1000,1/800, 1/700, 1/600, 1/500, 1/400, 1/250, 1/200, 1/150, 1/100, 1/75,1/50, 1/25, 1/10, 1/7, ⅕, ½, or 1 times the rate of diffusivity of theparticle in water under identical conditions. For example, at a timescale of 1 s, the rates of diffusion of unmodified or referenceparticles can be slower in brain tissue than the same particles inwater.

The density of coating of PEG or other material can affect the diffusionof nanoparticle within brain parenchyma. In some embodiments the MSD at1 sec of densely PEGylated particles is at least 5-fold greater thanthat of less-densely PEGylated particles, or at least 29-fold higherthan that of non-PEGylated particles. In further embodiments, the meansquare displacement (MSD) at 1 sec of densely PEGylated particles isonly 260-fold or less slower in brain tissue than in artificial cerebralspinal fluid (aCSF), whereas less-densely PEGylated particles can be upto 930-fold slower and non-PEGylated particles can be up to 6,900-foldslower. In a particular embodiment, at least 63% of densely PEGylatednanoparticles are capable of movement through rat brain parenchyma,relative to 32.9% of less-densely PEGylated particles and 10.3% ofnon-PEGylated particles, respectively.

The heterogeneity in particle transport rates can also be evaluated byexamining the distribution of individual particle diffusivities over aparticular time period, e.g., 1 s. In one embodiment, at least 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or greaterof coated particles of a given average particle size are classified asdiffusive.

ii. Electro-Kinetic Potential

The presence of the PEG or coating agent can affect the zeta-potentialof the particle. In one embodiment, the zeta potential of the particlesis between −10 mV and 100 mV, between −10 and 50 mV, between −10 mV and25 mV, between −5 mV and 20 mV, between −10 mV and 10 mV, between −10 mVand 5 mV, between −5 mV and 5 mV, or between −2 mV and 2 mV. In apreferred embodiment, the surface charge is near neutral.

iii. Particle Size

In some embodiments, the disclosed nanoparticles have an averagediameter equal to or smaller than the pores in the ECM of the brain. Inparticular embodiments, the particles have an average diameter fromabout 40 nm up to about 150 nm, up to about 100 nm, or up to about 60nm, more preferably about 50 nm. Particle size can be measured using anytechnique known in the art, for example using dynamic light scattering.Particle size may also be referenced with respect to a population,wherein a percentage of 60, 65, 70, 75, 80, 85, 90, 95% of the particleshave diameters within the specified range.

In another embodiment, the particles have an average diameter such thata majority of the particles do not become localized within cells ormicro-domains within tissue compared to larger particles. As shown inthe Table 1, particles having an average particle size of 50 nm showed alarger MSD at 1 sec when densely PEGylated, as measured using multipleparticle tracking (MPT) of fluorescently labeled gene vectors in rodentbrain.

In certain embodiments the nanoparticles release an effective amount ofthe nucleic acids over a period of at least 10 minutes, 20 minutes, 30minutes, one hour, two hours, hour hours, six hours, ten hours, one day,three days, seven days, ten days, two weeks, one month, or longer.

iv. Toxicity

The disclosed nanoparticles densely-coated with PEG or other coatingagents are less toxic than non-coated or conventionally coatedparticles. The in vitro or in vivo toxicity of nanoparticles can beassessed using any technique known in the art, such as cell viabilityassays. In some embodiments, toxicity of the particles is associatedwith the ratio of polymer to DNA. For example, a ratio of polymer to DNAof 1, or 2, or 3, or 4, can be less toxic than a ratio of polymer to DNAgreater than 4, such as a ratio of 5 or more.

The toxicity of the nanoparticles can be dependent upon the cell-type ortissue-type and can depend upon the concentration of the nanoparticles.In some embodiments toxicity is considered low when 75% of normalprimary cells are viable following exposure to nanoparticles at aconcentration of 10 μg/ml for two hours.

B. Pharmaceutical Excipients for Delivery to the Brain

The particles may be administered in combination with a physiologicallyor pharmaceutically acceptable carrier, excipient, or stabilizer.Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. Inpreferred embodiments, the particles are formulated for parenteraldelivery to the brain. Typically the particles will be formulated insterile saline or buffered solution for injection into the tissues orcells to be treated. The particles can be stored lyophilized in singleuse vials for rehydration immediately before use. Other means forrehydration and administration are known to those skilled in the art.

Optional pharmaceutically acceptable excipients include, but are notlimited to, stabilizers and surfactants. Stabilizers are used to inhibitor retard decomposition reactions which include, by way of example,oxidative reactions.

The nanoparticles or nanoconjugates can be formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof conjugate appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions willbe decided by the attending physician within the scope of sound medicaljudgment. For any nanoparticle or nanoconjugate, the therapeuticallyeffective dose can be estimated initially either in cell culture assaysor in animal models, usually mice, rabbits, dogs, or pigs. The animalmodel is also used to achieve a desirable concentration range and routeof administration. Such information can then be used to determine usefuldoses and routes for administration in humans. Therapeutic efficacy andtoxicity of conjugates can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose is therapeutically effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies can be used in formulating a range of dosagesfor human use.

II. Methods of Manufacture

A. Polymer Preparation

The polymers can be synthesized by any means known in the art. PEG orother coating agents can be conjugated to the core polymer using avariety of techniques known in the art depending on whether the coatingis covalently or non-covalently associated with the particles.

In some embodiments the PEG or other coating agent can be covalentlyattached to the core polymer by reacting functional groups on theparticles with reactive functional groups on the PEG or other coatingagent to make a copolymer. For example, aminated PEG can be reacted withreactive functional groups on the particles, such as carboxylic acidgroups, to covalently attach the agent via an amide bond.

In one embodiment methoxy-PEG-NHS is conjugated to 25 kDa branched PEIto yield a PEG-PEI copolymer. The extent of PEGylation of the resultingPEI copolymer can be varied by varying the molar ratio of PEG added tothe PEI.

In some embodiments nanoparticles are formed of a mixture of PEGylatedand non-PEGylated polymers. The non-PEGylated polymers can contribute adefined amount of the total free amines, such as 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50% or more than 50% of the total free amines in theparticles.

B. Nanoparticles

The disclosed nanoparticle gene carriers can be formed from one or morecationic polymers, one or more PEGs or other coating agents, and one ormore nucleic acids using any suitable method for the formation ofpolymer nanoparticles known in the art. The methods employed fornanoparticle formation will depend on a variety of factors, includingthe characteristics of the polymers present in the nanoparticle genecarrier, as well as the desired particle size and size distribution.

In circumstances where a monodisperse population of particles isdesired, the particles may be formed using a method which produces amonodisperse population of nanoparticles. Alternatively, methodsproducing polydisperse nanoparticle distributions can be used, and theparticles can be separated using methods known in the art, such assieving, following particle formation to provide a population ofparticles having the desired average particle size and particle sizedistribution.

Common techniques for preparing nanoparticles gene carriers include, butare not limited to, solvent evaporation, solvent removal, spray drying,phase inversion, low temperature casting, and nanoprecipitation.Suitable methods of particle formulation are briefly described below.Pharmaceutically acceptable excipients, including pH modifying agents,disintegrants, preservatives, and antioxidants, can optionally beincorporated into the particles during particle formation. As describedabove, one or more additional active agents can also be incorporatedinto the nanoparticle gene carrier during particle formation.

1. Solvent Evaporation

In this method the polymer is dissolved in a volatile organic solvent,such as methylene chloride. Nucleic acid is added to the solution, andthe mixture is suspended in an aqueous solution that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent evaporated, leaving solidnanoparticles. The resulting nanoparticles are washed with water anddried overnight in a lyophilizer. Nanoparticles with different sizes andmorphologies can be obtained by this method. This method is useful forrelatively stable polymers like polyesters and polystyrene.

However, labile polymers, such as polyanhydrides, may degrade during thefabrication process due to the presence of water. For these polymers,the following two methods, which are performed in completely anhydrousorganic solvents, are more useful.

2. Solvent Removal

This technique is primarily designed for polyanhydrides. In this method,the drug is dispersed or dissolved in a solution of the selected polymerin a volatile organic solvent like methylene chloride. This mixture issuspended by stirring in an organic oil (such as silicon oil) to form anemulsion. Unlike solvent evaporation, this method can be used to makenanoparticles from polymers with high melting points and differentmolecular weights. The external morphology of spheres produced with thistechnique is highly dependent on the type of polymer used.

3. Spray-Drying

In this method, the polymer is dissolved in organic solvent. A knownamount of the active drug is suspended (insoluble drugs) or co-dissolved(soluble drugs) in the polymer solution. The solution or the dispersionis then spray-dried.

4. Phase Inversion

Microspheres can be formed from polymers using a phase inversion methodwherein a polymer is dissolved in a “good” solvent, fine particles of asubstance to be incorporated, such as a drug, are mixed or dissolved inthe polymer solution, and the mixture is poured into a strong nonsolvent for the polymer, to spontaneously produce, under favorableconditions, polymeric microspheres, wherein the polymer is either coatedwith the particles or the particles are dispersed in the polymer. Themethod can be used to produce nanoparticles in a wide range of sizes,including, for example, about 100 nanometers to about 10 microns.Exemplary polymers which can be used include polyvinylphenol andpolylactic acid. Substances which can be incorporated include, forexample, imaging agents such as fluorescent dyes, or biologically activemolecules such as proteins or nucleic acids. In the process, the polymeris dissolved in an organic solvent and then contacted with anon-solvent, which causes phase inversion of the dissolved polymer toform small spherical particles, with a narrow size distributionoptionally incorporating an antigen or other substance.

Other methods known in the art that can be used to prepare nanoparticlesinclude, but are not limited to, polyelectrolyte condensation (see Suket al., Biomaterials, 27, 5143-5150 (2006)); single and double emulsion(probe sonication); nanoparticle molding, and electrostaticself-assembly (e.g., polyethylene imine-DNA or liposomes).

III. Methods of Use

It has been established that the density and composition of a surfacecoating agent such as PEG can determine the ability of the particles todiffuse throughout the brain parenchyma. The diffusion limitations ofnanoparticles (˜50 nm diameter particles) was investigated ex vivo, inexcised rodent brain slices, as described in the Examples. Usingmultiple particle tracking (MPT) and optimized PEGylation protocols, itwas shown that differences in PEG coating density and molecular weighthave a significant impact on shielding particles from adhesiveinteractions and enabling them to penetrate and distribute moreuniformly in vivo.

Therefore, the particle compositions described herein can be used toadminister one or more therapeutic, prophylactic, and/or diagnosticagents directly to the brain to treat one or more diseases or disordersof the brain.

A. Therapeutic Uses

Nanoparticle gene carriers carrying one or more nucleic acid can beutilized to deliver nucleic acid cargo for therapeutic or prophylacticpurposes, such as in a method of gene therapy. Methods of gene therapytypically rely on the introduction into the cell of a nucleic acidmolecule that alters the genotype of the cell. Introduction of thenucleic acid molecule can correct, replace, or otherwise alter theendogenous gene via genetic recombination. Methods can includeintroduction of an entire replacement copy of a defective gene, aheterologous gene, or a small nucleic acid molecule such as anoligonucleotide. For example, corrective gene can be introduced into anon-specific location within the host's genome. This approach typicallyrequires delivery systems to introduce the replacement gene into thecell, such as genetically engineered viral vectors.

1. Disorders or Diseases to be Treated

Exemplary diseases and disorders of the brain that can be treated by thedisclosed compositions and methods include neoplasms (cancers, tumors,growths), infections (HIV/AIDS, Tuberculosis), inflammation (multiplesclerosis, transverse myelitis and other autoimmune processes, cerebralor tissue edema and other reactive processes), acquired or degenerativeconditions (Alzheimer's disease, Parkinson's disease, stroke,amylotrophic lateral sclerosis, acute and chronic traumatic and painsyndromes), congenital or genetic abnormalities (neurofibromatosis,mucopolysaccaridoses, tuberous sclerosis, Von Hippel Lindau), epigeneticconditions and brain trauma or injury.

B. Methods of Administration and Dosing

The disclosed nanoparticles can be administered by a variety of routesof administration. In certain embodiments the particles are administereddirectly to the brain. In other embodiments the particles areadministered systemically.

The composition of the brain ECM, including the physico-chemicalproperties of its components and the space between them (‘pores’), arekey factors that determine the penetration of substances within thebrain.

Unshielded, negatively charged particles with exposed hydrophobicregions have significantly hindered diffusion regardless of particlesize. The hydrophobic interactions between particle surfaces and ECMcomponents can be a source of significant adhesion. Adequate surfaceshielding from potential interactions, including electrostatic andhydrophobic forces, are crucial for rapid diffusion in the brain.

Mechanisms for the enhanced delivery of the disclosed gene vectors tothe brain are disclosed. Enhanced local delivery can be achieved viaconvection, electricomagnetic, or other forces Enhanced systemicdelivery can be achieved via co- or sequential administration withpermeabliization agents such as but not limited to pharmacologicsubstances (e.g. cytokines), mechanical barrier disruption (e.g.ultrasound), or osmotic changes (e.g. mannitol). Other methods ofdelivery include intrathecal or intra-ventricular delivery viacerebro-spinal fluid spaces, intra-nasal administration or delivery viathe olfactory bulb and systemic delivery via oral, intravenous, orintra-arterial administration.

1. Convection Enhanced Delivery

In some embodiments the brain penetrating capability of the disclosednanoparticles is enhanced following convection enhanced delivery (CED).CED is a method in which drugs are delivered through a needle installedintraparenchymally into the brain and attached to a pump providingpositive pressure and constant flow of the infusates. For example,densely PEGylated nanoparticles drugs can be delivered through one toseveral catheters placed stereotactically, for example, directly withina brain tumor mass or around the tumor or the resection cavity.

In some embodiments CED can significantly enhances distribution ofvaried-size molecules and increase the infused compounds' locoregionalconcentration. In certain embodiments the use of CED to deliver denselyPEGylated particles enhances the distribution of the particlesthroughout the brain to an extent that is greater than expected. In someembodiments gene vector distribution and high-level transgene expressioncan be achieved throughout the entire striatum. CED is unlikely toprovide a significant benefit if particles, such as the referenceparticles, remain entrapped in the brain parenchyma due to adhesiveinteractions and/or steric obstruction. Thus, physicochemical propertiesof particles that allow unhindered diffusion in the brain parenchymaremain critical for achieving enhanced particle penetration followingthe CED.

2. Administration Regimes

In general the timing and frequency of administration will be adjustedto balance the efficacy of a given treatment or diagnostic schedule withthe side-effects of the given delivery system. Exemplary dosingfrequencies include continuous infusion, single and multipleadministrations such as hourly, daily, weekly, monthly or yearly dosing.

Regardless of systemic, intrathecal, or local delivery into the brainparenchyma itself, penetration of bioactive or imaging agents in thebrain and other tissues has been a key hurdle to effective therapy anddiagnostics. Numerous studies using viral, nanoparticle, andconvection-enhanced delivery have failed due to limited movement ofsubstances within the brain. Therefore, defining the critical limitingparameters and designing strategies to enhance brain penetration willlikely improve the efficacy of these treatments. Densely-PEGylatednanoparticles offer numerous additional advantages, including increasedparticle diffusion, improved stability, and prolonged sustained-releasekinetics. These factors are known to correlate with the efficacy of manytherapeutics and will likely have a significant impact on the utility ofnano-sized carriers for diagnostic and therapeutic delivery to thebrain.

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1: Preparation of Vectors to Shield the Positive Surface ChargeIntrinsic to Cationic Polymer-Based Gene Vectors

Materials and Methods

Polymer Preparation

Methoxy PEG N-hydroxysuccinimide (mPEG-NHS, 5 kDa, Sigma-Aldrich, St.Louis, Mo.) was conjugated to 25 kDa branched polyethyleneimine (PEI)(Sigma-Aldrich, St. Louis, Mo.) to yield a PEG5k-PEI copolymer aspreviously described [30]. Briefly, PEI was dissolved in ultrapuredistilled water, the pH was adjusted to 7.5-8.0 and mPEG-NHS was addedto the PEI solution at various molar ratios and allowed to reactovernight in 4° C. The polymer solution was extensively dialyzed (20,000MWCO, Spectrum Laboratories, Inc., Rancho Dominguez, Calif.) againstultrapure distilled water and lyophilized. Nuclear magnetic resonance(NMR) was used to confirm a PEG:PEI ratio of 8, 26, 37 and 50. 1H NMR(500 MHz, D2O): δ 2.48-3.20 (br, CH2CH2NH), 3.62-3.72 (br, CH2CH2O). Thepoly-L-lysine 30-mer (PLL) and PEGSK-PLL block copolymers weresynthesized and characterized as previously published [Suk, J. S., etal. J Control Release, 2014; Kim, A. J., et al., J Control Release,2012. 158(1): p. 102-7.]. The lyophilized polymers were dissolved inultrapure distilled water and pH was adjusted to ˜6.5-7.

Gene Vector Complexation

The pd1GL3-RL plasmid DNA was a kind gift from Professor Alexander M.Klibanov (M.I.T) and pEGFP plasmid was purchased by ClontechLaboratories Inc. (Mountainview, Calif.). The plasmid DNA was propagatedand purified as previously described [Suk, J. S., et al. J ControlRelease, 2014]. Mirus Label IT® Tracker™ Intracellular Nucleic AcidLocalization Kit (Mirus Bio, Madison, Wis.) was used to fluorescentlytag plasmid DNA with a Cy3 or Cy5 fluorophore. Gene vectors were formedby the drop-wise addition of 10 volumes of labeled or non-labeledplasmid DNA (0.2 mg/ml) to 1 volume of a swirling polymer solution. PEIsolutions were prepared at previously optimized nitrogen to phosphate(N/P) ratio of 6 and at PEG5k-PEI to PEI molar ratio of 3. For theformulation of free PEI and (PEG5k)8-PEI based gene vector controls, thePEI solutions were prepared at N/P ratio of 6 using 100% of free PEI or(PEG5k)8-PEI, respectively. For fluorescence imaging, Cy3- orCy5-labeled DNA was used to assemble fluorescently labeled gene vectors.The plasmid/polymer solutions were incubated for 30 min at roomtemperature to form gene vectors. Gene vectors were washed twice with 3volumes of ultrapure distilled water, and re-concentrated to 1 mg/mlusing Amicon® Ultra Centrifugal Filters (100,000 MWCO, Millipore Corp.,Billerica, Mass.) to remove free polymers. DNA concentration wasdetermined via absorbance at 260 nm using a NanoDrop ND-1000spectrophotometer (Nanodrop Technologies, Wilmington, Del.). PEG-PLLnanoparticles were similarly prepared at an N/P ratio of 2 as previouslydescribed [Suk, J. S., et al. J Control Release, 2014; Kim, A. J., etal., J Control Release, 2012. 158(1): p. 102-7; Boylan, N.J., et al.,Biomaterials, 2012. 33(7): p. 2361-71.].

Physicochemical Characterization of Nanoparticles

Hydrodynamic diameter, ζ-potential and polydispersity were measured in10 mM NaCl at pH 7.0 by dynamic light scattering and laser Doppleranemometry, respectively, using a Nanosizer ZS90 (Malvern Instruments,Southborough, Mass.). Gene vectors were imaged using transmissionelectron microscopy (TEM, Hitachi H7600, Japan) to determine theirmorphology and size.

Results

To effectively shield the positive surface charge intrinsic to cationicgene vectors, gene vectors using copolymers of multiple 5 kDa PEGmolecules conjugated to PEI (PEG5k-PEI) were formulated with a range ofPEG to PEI molar ratios. As previously reported, PEGylation of cationicpolymers may have negative influences on DNA complexation due toreduction of available positive charges resulting from the PEGconjugation and additional steric hindrance imposed by grafted PEGchains [Petersen, H., et al., Bioconjug Chem, 2002. 13(4): p. 845-54].Thus, the conventional DNA complexation method, using only highlyPEGylated PEI copolymers, yields loose, unstable DNA nanoparticles whichare not likely to retain their stability in biological specimens. Inorder to achieve compact and colloidally stable gene vectors, vectorswere formulated with a blend of PEG5k-PEI and free PEI with 25% ofamines deriving from free PEI, as described by Suk, J. S., et al. JControl Release, 2014. Using a fixed amount of free PEI, the compactionof DNA in ˜50 nm particles was achieved using PEG5k-PEI copolymers witha wide range of PEG to PEI molar ratios (Table 2). The use of acopolymer with a PEG to PEI ratio of 26, which is substantially higherthan conventionally used PEGylation ratios [Petersen, H., et al.,Bioconjug Chem, 2002. 13(4): p. 845-54; Malek, et al., J Drug Target,2008. 16(2): p. 124-39; Merkel, et al., Biomaterials, 2011. 32(21): p.4936-42], is sufficient to form gene vectors with a near neutralζ-potential (Table 1) and potentially allows for brain penetration(brain penetrating nanoparticle; BPN hereafter). In the subsequentstudies the BPN was compared to similarly sized conventionally PEGylatednanoparticles (CPN), consisting of PEGylated PEI with a lower PEG to PEIratio of 8 [Petersen, et al., Bioconjug Chem, 2002. 13(4): p. 845-54;Malek, et al., Toxicol Appl Pharmacol, 2009. 236(1): p. 97-108; Lutz, etal., Methods Mol Biol, 2008. 433: p. 141-58.], and un-PEGylated PEInanoparticles (UPN). The physicochemical properties of BPN, CPN and UPNare summarized in Table 1. Of note, CPN possessed larger particlediameter and more positive surface charge compared to BPN, suggestingthe looser compaction and/or inferior surface coating.

TABLE 1 Physicochemical properties and diffusivity of gene vectors inrodent cortical tissue. Hydrodynmc Hydrodynmc Diameter ± ζ-potential ±Diameter in MSD_(AQ)/ SEM (nm) SEM (mV) PDI ACSF MSD_(Brain) UPN 47 ± 2 26 ± 1.2 0.15 392 ± 8 6900 CPN 59 ± 1 9.3 ± 0.5 0.17 172 ± 2 930 BPN 43± 5 2.9 ± 0.3 0.19  50 ± 9 260

Size, ζ-potential and polydispersity (PDI) were measured by dynamiclight scattering (DLS) in 10 mM NaCl at pH 7.0 and are presented asaverage of at least 3 measurements±standard error (SEM). Mean squaredisplacement (MSD) at 1 sec was measured using multiple particletracking (MPT) of fluorescently labeled gene vectors in rodent brainslice. NP diffusivity in aCSF was calculated using the Stokes-Einsteinequation and the mean particle diameter. Hydrodynamic diameter in aCSFwas measured by DLS following incubation in aCSF at 37° C. for 1 hour.UPN: un-PEGylated Nanoparticles, CPN: Conventionally PEGylatedNanoparticles, BPN: Brain Penetrating Nanoparticles.

TABLE 2 Physicochemical properties and diffusivity of PEI-based genevectors in rodent cortical tissue. Hydrodynmic Diameter ± ζ-potential ±SEM (nm) SEM (mV) PDI (PEG_(5k))₈-PEI 55 ± 4 6.7 ± 0.7 0.17(PEG_(5k))₂₆-PEI 43 ± 5 2.9 ± 0.3 0.19 (PEG_(5k))₃₇-PEI 44 ± 1 3.4 ± 0.50.17 (PEG_(5k))₅₀-PEI 48 ± 3 1.8 ± 0.8 0.14

Size, ζ-potential and polydispersity (PDI) were measured by dynamiclight scattering (DLS) in 10 mM NaCl at pH 7.0 and are presented asaverage of at least 3 measurements±standard error (SEM).

Nanoparticle diffusion in the brain predominantly takes place throughthe narrow tortuous space between cells [Sykova, E. and C. Nicholson,Physiol Rev, 2008. 88(4): p. 1277-340]. The ECM, the main component ofthe extracellular space, imposes an adhesive and steric barrier to themovement of nanoparticles through the brain parenchyma. Nonspecificelectrostatic interactions with the abundant negative charges of the ECMhinder the diffusion of poorly shielded cationic polymer-based genevectors [Zimmermann, et al., Histochem Cell Biol, 2008. 130(4): p.635-53; Ruoslahti, Glycobiology, 1996. 6(5): p. 489-92], as shown withUPN and CPN in this study. Hence, rapid brain penetration of BPN is mostlikely attributed to the efficient shielding of this positive surfacecharge intrinsic to the cationic polymer-based gene vectors. Moreover,the dense surface PEG coating enabled by the blending technique allowsBPN to retain their compact sub-100 nm size in physiological conditions(i.e. CSF) required to move through the ECM mesh pores without beinghindered by steric obstruction. In comparison, the loose compaction,lack of stability and the tendency towards aggregation of conventionallyPEGylated cationic particles, including CPN, does not allow forefficient penetration through the ECM which has pore sizes smaller than200 nm [Nance, E. A., et al., Sci Transl Med, 2012. 4(149): p. 149ra119,MacKay, et al. Brain Res, 2005. 1035(2): p. 139-53]. These results,demonstrate the importance of designing gene vectors capable ofovercoming both the adhesive interactions and steric hindrance imposedby the brain ECM.

Example 2: Gene Vector Particles are Stable Following Incubation inPhysiological Environment

Materials and Methods

PEI nanoparticle stability was assessed by incubating nanoparticles inartificial cerebrospinal fluid (aCSF; Harvard Apparatus, Holliston,Mass.) at 37° C. and conducting dynamic light scattering every 30 minsfor 24 hours. After 1 hour of incubation, a fraction of the nanoparticlesolution was removed and imaged using TEM.

Results

To predict the particle stability of gene vectors following in vivoadministration, the in vitro stability in artificial cerebrospinal fluid(aCSF) was characterized over time at 37° C. (FIG. 1). UPN aggregatedimmediately after adding in aCSF. In 1 hour, the hydrodynamic diameterincreased 8.3-fold and in 7 hours, the polydispersity was larger than0.5, indicating loss of colloidal stability. CPN increased in diameterby 3-fold following incubation in aCSF and remained stable over 24hours. BPN, formulated by the blend approach, exhibited improvedstability in aCSF compared to both UPN and CPN. BPN remained unchangedover the first 1 hour followed by a 2 fold increase in diameter whichremained stable over 24 hours (Table 1; FIG. 1). These results werefurther confirmed by transmission electron micrographs of gene vectorsin ultrapure water and after 1 hour incubation in aCSF at 37° C.Incubation of UPN in aCSF resulted in the formation of large aggregates.BPN and CPN qualitatively increased in size but retained theirintegrity.

Example 3: Gene Vector Particles are Non-Toxic In Vitro and In Vivo

Materials and Methods

Cell Culture

9 L gliosarcoma cells were provided by Dr. Henry Brem. 9 L immortalizedcells were cultured in Dulbecco's modified Eagle's medium (DMEM,Invitrogen Corp., Carlsbad, Calif.) supplemented with 1%penicillin/streptomycin (pen/strep, Invitrogen Corp., Carlsbad, Calif.)and 10% heat inactivated fetal bovine serum (FBS, Invitrogen Corp.,Carlsbad, Calif.). When cells were 70-80% confluent, they were reseededin 96-well plates to assess toxicity and in 24-well plates to assesstransfection and cell uptake of gene vectors. Rabbit primary astrocyteswere provided by Dr. Sujatha Kannan. Mixed cell culture was preparedfrom day 1 neonatal rabbits and astrocytes were isolated using theconventional shake off method. Astrocytes were cultured in DMEMsupplemented with 1% pen/strep and 10% FBS and passaged once; when cellswere 70-80% confluent they were reseeded in 96-well plates for cellviability assay. Rat brain primary astrocytes were provided by Dr. ArunVenkatesan. Rat brain primary mixed cultures were isolated form neonatalP3-P6 rats and astrocytes were isolated with the conventional shake offmethod as previously published [Hosmane, et al., Journal ofNeuroscience, 2012. 32(22): p. 7745-7757]. Cells were cultured inDMEM/F12 (Invitrogen Corp., Carlsbad, Calif.) supplemented with 10% FBSand 1% pen/strep. When cells were 70-80% confluent on passage one, theywere immediately reseeded in 96-well plates to assess gene vectortoxicity and in 24-well plates to assess transfection and cell uptake ofgene vectors.

In Vitro Toxicity

Cells were seeded onto 96-well plates at an initial density of 1.0×10⁴cells/well and incubated at 37° C. After 24 h, cells were incubated witha wide range of doses of DNA nanoparticles in media for 24 h at 37° C.Cell viability was assessed using the Dojindo cell counting kit-8(Dojindo Molecular Technologies, Inc., Rockville, Md.). Absorbance at450 nm was measured spectrophotometrically using the Synergy MxMulti-Mode Microplate Reader (Biotek, Instruments Inc.).

Results

Despite their wide use as non-viral gene vector systems, PEI-based genevectors have raised concerns of their toxicity due to their highpositive charge density [Petersen, et al., Bioconjug Chem, 2002. 13(4):p. 845-54; Merkel, et al., Biomaterials, 2011. 32(21): p. 4936-42]. Toensure their safety for administration to the CNS, the in vitro toxicitymediated by the gene vectors was thoroughly characterized in primaryastrocytes derived from neonatal rabbits (FIG. 2A), primary astrocytesderived from rats (FIG. 2B) and 9 L rat gliosarcoma cells (FIG. 2C)using plasmid concentrations from 1 to 10 μg/ml. Both UPN and CPNexhibited cytotoxicity in all three cells tested; UPN resulted in 50%cell death at 5, 10, and 10 μg/ml of plasmid concentration for primaryrabbit cells, primary rat cells and 9 L cells, respectively. Similarly,CPN resulted in less than 50% cell viability at 10 μg/ml for primaryrabbit and primary rat astrocytes. CPN treatment of 9 L gliosarcomacells at 5 and 10 μg/ml of plasmid resulted in approximately 70% cellviability.

Contrary to these findings, the BPN were non-toxic in rabbit primaryastrocytes and 9 L gliosarcoma cells and showed only mild toxicity inrat primary astrocytes even at a high concentration of 10 μg/ml (FIGS.2A-2D). the toxicity of BPN was compared to a PEG-PLL nanoparticlesystem shown to be safe in animals [Yurek, et al., Cell Transplant,2009. 18(10): 1183-1196; Yurek, et al., Mol Ther, 2009. 17(4): 641-650]and humans [Konstan, et al., Hum Gene Ther, 2004. 15(12): 1255-1269].BPN and PEG-PLL exhibited similar safety profiles in all three celltypes at varying concentrations. In summary, conventional PEG coatingdoes not sufficiently reduce cytotoxicity [Davies, et al., Mol Ther,2008. 16(7): 1283-1290]. However, BPN demonstrate favorable safetyprofiles even at high plasmid doses. The in vivo safety profile of thesegene vectors were further histopathologically characterized, followingCED. In accordance to these in vitro data, UPN demonstrated higher signsof toxicity than CPN and BPN. BPN and CPN demonstrated no toxicity astheir effect did not differ from that of normal saline administration(FIG. 2D). Importantly, regardless of the gene vector type, inflammationand hemorrhage was confined around the injection site and did notpropagate through the brain tissue.

Cytotoxicity of cationic polymer-based gene vectors has long beenacknowledged as a limitation for the use of these versatile and potentgene delivery platforms [Petersen, et al., Bioconjug Chem, 2002. 13(4):845-854; Merkel, et al., Biomaterials, 2011. 32(21): 4936-4942.]. Ingood agreement with previous observations [Petersen, et al., BioconjugChem, 2002. 13(4): 845-854; Davies, et al., Mol Ther, 2008. 16(7):128312-90, Beyerle, et al., Toxicol Appl Pharmacol, 2010. 242(2):146-154], conventional PEGylation (i.e. CPN) is not sufficient tosignificantly improve the in vitro safety profile of cationicpolymer-based gene vectors. Dense PEGylation achieved by the blendapproach drastically decreases the toxicity of BPN, leading to afavorable safety profile similar to the widely used PEG-PLL nanoparticlesystem shown to be safe in animals [Yurek, et al., Cell Transplant,2009. 18(10): 1183-1196; Yurek, et al., Mol Ther, 2009. 17(4): 641-650]and humans [Konstan, et al., Hum Gene Ther, 2004. 15(12): 1255-1269.].

Example 4: PEGylated Nanoparticle Gene Vectors have High Uptake andTransfection Efficiencies

Materials and Methods

In Vitro Transfection

Cells were seeded onto 24-well plates at an initial density of 5.0×10⁴cells/well. After 24 h, cells were incubated with pd1GL3-RL plasmid ingene vector form (1 μg DNA/well) in media for 5 h at 37° C. Cationicpolymer-based gene vector transfection was compared to free plasmidcontrol. Subsequently, nanoparticles and culture media were replacedwith fresh media. After additional 48 h of incubation at 37° C., mediawas removed and 0.5 ml of 1× Reporter Lysis Buffer was added. Cells weresubjected to three freeze-and-thaw cycles to assure complete cell lysis,and supernatants were obtained by centrifugation. Luciferase activity inthe supernatant was then measured using a standard luciferase assay kit(Promega, Madison, Wis.) and a 20/20 n luminometer (Turner Biosystems,Sunnyvale, Calif.). The relative light unit (RLU) was normalized to thetotal protein concentration of each well measured by Bio-Rad proteinassay.

Results

Cell uptake and transfection efficiencies of the gene vectors werecharacterized in vitro. PEG coating has been shown to reduce particleuptake by cells [Amoozgar, Z. and Y. Yeo, Wiley Interdiscip Rev NanomedNanobiotechnol, 2012. 4(2): 219-233; Hatakeyama, et al. Adv Drug DelivRev, 2011. 63(3): 152-160]. However, in good agreement with previousreports [Mishra, et al., Eur J Cell Biol, 2004. 83(3): 97-111.],conventional PEGylation did not affect cell uptake by PEI-based genevectors (i.e. CPN). The small particle size may contribute to effectiveuptake of PEGylated nanoparticles [Pamujula, et al., J Pharm Pharmacol,2012. 64(1): 61-67; Hu, Y., et al., J Control Release, 2007. 118(1):7-17]. BPN, despite their denser PEG coating compared to CPN, alsopresented no difference in uptake compared to UPN and CPN. All threePEI-based gene vectors were detected in 50% of 9 L immortalized cells(FIG. 3A) and 35% of rodent primary cells (FIG. 3B). The uptake of thesegene vectors was ˜17 and ˜35 fold higher than clinically tested PEG-PLLgene vectors in 9 L immortalized and primary astrocytes, respectively(p<0.05). This difference translated to a significantly higherluciferase expression by PEI-vector treated cells in comparison to celltreated with PEG-PLL vector at the same plasmid dose. Despite thesimilar cell uptake among different PEI-based gene vectors,significantly lower in vitro transgene expression by both CPN and BPNwas found compared to UPN (FIGS. 3C and 3D), in accordance with previousobservations [Mishra, et al., Eur J Cell Biol, 2004. 83(3): 97-111].

Example 5: BPN PEGylated Nanoparticle Gene Vectors have RapidlyPenetrate the Brain Parenchyma

Materials and Methods

Animal Studies

Female Fischer 344 rats, weighing 120-140 g each, were purchased fromHarlan Laboratories (Frederick, Md.). The use of inbred rats waspreferred to other outbred strains due to the radical impact of geneticdifferences in gene expression [Liu, et al., J Biol Chem, 2002. 277(7):4966-4972]. They were housed in standard facilities and given freeaccess to food and water. All animals were treated in accordance withthe policies and guidelines of the Johns Hopkins University Animal Careand Use Committee. All surgical procedures were performed usingstandard, sterile surgical technique.

Rats were anesthetized with a mixture of ketamine-xylazine as previouslydescribed [Recinos, et al., Neurosurgery, 2010. 66(3): 530-537;discussion 537]. Briefly, 350 μL of a ketamine (75 mg/kg), xylazine (7.5mg/kg), ethanol (14.25%), and 0.9% normal saline solution wasadministered intraperitoneally. A midline scalp incision was made toexpose the coronal and sagittal sutures and a burr whole was drilled 3mm lateral to the saggital suture and 0.5 mm posterior to the bregma.Following the administration of nanoparticle solution the skin wasclosed using biodegradable sutures (POLYSORB™ Braided Absorbable Sutures5-0) and Bacitracin was applied.

To study the diffusion based spread of nanoparticles in vivo, N=3animals were used; a 33 gauge 10 μl Hamilton Neuro Syringe mounted to astereotaxic headframe was lowered to a depth of 3.5 mm and retracted 1mm to create a pocket in the rodent striatum in order to minimize theconvective flow during infusion. A 10 μl solution of Cy5 labeledconventionally PEGylated nanoparticles and Cy3 labeled brain penetratingnanoparticles at a plasmid concentration of 500 μg/ml per particle typewas administered as a bolus injection at 2 μl/min Animals weresacrificed 2 hours following the injection.

To study the distribution of PEI based gene vectors following convectionenhanced delivery in the rodent striatum N=6 rats were used; A 33 gauge50 μl Hamilton Neuro Syringe mounted to a stereotaxic headframe waslowered to a depth of 3.5 mm. A 20 μl solution of Cy3 labeled CPNs andCy5 labeled BPNs at a plasmid concentration of 500 μg/ml per particletype in normal saline was administered. The rate of infusion was set at0.33 μl/min, using a Chemyx Inc. Nanojet Stereotaxic syringe pump(Chemyx, Stafford, Tex.). Animals were sacrificed 5 hours following theinjection. To examine the dependence of distribution on nanoparticleconcentration co-injections were also performed at half the plasmidconcentration, 250 μg/ml per particle type, in normal saline.

To assess the distribution of transgene expression following CEDadministration of gene vectors, at least N=4 rats per particle type wereused; Plasmid encoding fluorescent eGFP reporter protein with acytomegalovirus (CMV) promoter was complexed into the various PEI-basednanoparticle formulations and infused in a 20 μl solution of 1 mg/mlplasmid solution using the same parameters described above. Animals weresacrificed 48 hours following CED administration and harvested brainswere fixed in 4% formaldehyde.

For Western blot analysis of in vivo transfection following CED of genevectors, N=3 rats per particle type were used and the exact sameexperimental procedures followed for imaging based analysis ofdistribution of transfection were used. Animals were sacrificed 48 hoursfollowing CED administration and immediately placed on ice and a 4 mmthick coronal slice of the striatum from −2 mm to 2 mm from theinjection site was dissected and stored in −80° C. for Western blotanalysis.

To assess the safety profile of the gene vectors in vivo following CEDadministration, N=3 rats per group were used. Various PEI-basedformulations were infused in in a 20 μl solution at a 1 mg/ml plasmidconcentration as described above. A normal saline solution was infusedas a negative control for comparison. Animals were sacrificed 4 daysfollowing administration and the harvested brains were fixed in 4%formaldehyde, processed, sectioned and stained with hematoxylin andeosin. Blind histopathological analysis was performed by a boardcertified neuropathologist and tissues were scored from 0-3 forindications of inflammation and hemorrhage (0: noinflammation/hemorrhage, 1: mild, 2: moderate, 3: severe).

Multiple Particle Tracking in Rodent Brain Slices

Multiple particle tracking (MPT) was used to estimate the mean squaredisplacement (MSD) of fluorescent gene vectors in ex vivo rodent brainslices as previously published [Nance, et al., Sci Transl Med, 2012.4(149): 149ra119]. Briefly, brain was harvested from adult Fisher ratsand incubated in aCSF for 10 minutes on ice. Brain was sliced into 1.5mm coronal slices using a Zivic brain matrix slicer (Zivic Instruments,Pittsburgh, Pa.) and placed on custom made slides. Half a microliter offluorescently labeled gene vectors was injected on the cerebral cortexat a depth of 1 mm using a 50 μl Hamilton Neuro Syringe (Hamilton, Reno,Nev.) mounted on a stereotaxic frame. Tissues were covered by a 22 mm×22mm coverslip to reduce tissue movement and bulk flow. Particletrajectories were recorded over 20 seconds at an exposure time of 66.7ms by a Evolve 512 EMCCD camera (Photometrics, Tucson, Ariz.) mounted onan inverted epifluorescence microscope (Axio Observer D1, Zeiss;Thornwood, N.Y.) equipped with a 100×/1.46 NA oil-immersion objective.Movies were analyzed with a custom made MATLAB code to extract x,y-coordinates of gene vectors centroids over time and calculate the meansquare displacement of each particle as a function of time [Nance, etal., Sci Transl Med, 2012. 4(149): p. 149ra119; Schuster, et al.,Biomaterials, 2013. 34(13): p. 3439-46]. The spatial resolution to thenoise to signal ratio correlation was estimated using immobilized genevectors on a glass slide [Martin, et al., Biophys J, 2002. 83(4):2109-17; Savin, et al., Biophys J, 2005. 88(1): 623-38]. Based on thatcorrelation the average resolution of the MPT experiments was estimatedto be ˜0.009 μm² at 1 second. At least N=3 rat brains were used per genevector type and at least 500 gene vectors were tracked per sample. Thegeometric mean of the MSDs for all nanoparticles was calculated persample and the average of different rodent brains was calculated as afunction of time. Histograms were generated from the MSD of everynanoparticle at a time scale of τ=1 sec. Theoretical MSD ofnanoparticles in ACSF was calculated using Stokes-Einstein equation andthe mean particle diameter calculated through dynamic light scattering.

Imaging and Analysis

Freshly harvested brains were fixed in 4% formaldehyde overnightfollowed by gradient sucrose solution processing before cryosection.Tissues were sectioned coronally into 100 micrometer thick slices usingLeica CM 1905 cryostat. Slices were stained with DAPI (Molecular Probes,Eugene, Oreg.) and imaged for DAPI (cell nuclei), Cy3 and Cy5 or AlexaFluor 488 (eGFP) using confocal LSM 710 microscope under 5× and 10×magnification (Carl Zeiss; Hertfordshire, UK). Settings were carefullyoptimized to avoid background fluorescence based on non-injected controlrat brains. Laser power, pinhole, gain, offset and digital gain wereselected separately for each magnification and kept constant throughoutthe study.

Statistical Analysis

Statistically significant differences between two groups were analyzedwith a two-tailed Student's t test assuming unequal variances or pairedstudent's t test when allowed. Multiple comparisons were performed usingone-way analysis of variance (ANOVA) followed by post hoc test usingSPSS 18.0 software (SPSS Inc. Chicago, Ill.).

Results

The diffusion of BPN, CPN and UPN in the brain parenchyma wasinvestigated. Due to their positive surface charge, the UPN werestrongly hindered with constrained non-Brownian time-lapse traces.Similarly, CPN exhibited less constrained but still hinderednon-Brownian motions. In contrast, BPN trajectories spanned over greaterdistances indicating the unhindered diffusion in brain tissue (FIG. 4A).Based on the trajectories, the ensemble averaged MSD (<MSD>) over 1second was calculated; BPN presented <MSD>5- and 29-fold higher than CPNand UPN, respectively (FIG. 4B). The diffusion rates of UPN and CPN inbrain tissue were 6,900- and 930-fold slower than their theoreticaldiffusion rates in aCSF, respectively, while BPNs moved only 260-foldslower in brain than in aCSF (Table 1). The individual particle data wasrepresented in a histogram of logarithmic MSD (log 10MSD) of individualgene vectors. The distribution was largely unimodal for UPN and BPN; themajority of UPN displayed low MSD values and most of the BPN showed MSDthat allowed rapid penetration in brain tissue. CPN were largely trappedbut a minor population was able to rapidly penetrate the brainparenchyma (FIG. 4C). Defining rapidly moving nanoparticles asnanoparticles with log 10MSD≥−1, 10.3%, 32.9% and 63% of UPN, CPN andBPN, respectively, were able to move in the brain parenchyma.

To test whether the enhanced brain penetration ex vivo by the BPNtranslated to wide spread of these vectors in brain parenchyma in vivo,a bolus co-injection of fluorescently labeled CPN and BPN was performedin the rodent striatum. Following the administration, CPN onlymoderately escaped from the injection site 2 hours after theadministration, whereas BPN homogeneously diffused farther away from theinjection site covering a distance of approximately 300 μm.

The high transfection and subsequent expression of therapeutic proteinsaway from the point of administration, constitutes the cornerstone forefficacious gene delivery-based treatment of CNS diseases. Effectivelycoating cationic polymer-based gene vectors allows for transgeneexpression over a larger volume of the brain striatum. However,PEGylation as a stealth coating strategy has been shown to decreaseuptake, endosome escape and subsequent transgene expression [Amoozgar,et al., Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2012. 4(2):219-33; Hatakeyama, et al., Adv Drug Deliv Rev, 2011. 63(3): 152-60].PEGylation of cationic polymer based gene vectors does not decrease cellentry, likely due to the small particle size, as suggested in previousreports [Pamujula, et al., J Pharm Pharmacol, 2012. 64(1): 61-7; Hu, etal., J Control Release, 2007. 118(1): 7-17], but leads to significantlylower transfection efficacy in vitro [Mishra, et al., Eur J Cell Biol,2004. 83(3): 97-111; Ogris, et al., AAPS PharmSci, 2001. 3(3): E21].This may be explained by the increased intracellular stability of BPNthat may hinder the DNA unpackaging and the conjugation of PEG toprimary amines that reduces buffering capacity of gene vectors and thesubsequent endosome escape [Mishra, et al., Eur J Cell Biol, 2004.83(3): 97-111; Sonawane, et al., J Biol Chem, 2003. 278(45): 44826-31].

Example 6: CED Acts with Gene Vector Physicochemical Properties toEnhance Distribution and Transgene Delivery of Dencely PEGylated GeneVectors

Materials and Methods

Imaging and Analysis

The nanoparticle volume of distribution following CED administration wasquantified by using a custom MATLAB script that subtracted thebackground fluorescence and thresholded the fluorescent intensities at10% of the maximum intensity. Nanoparticle fluorescence in the corpuscallosum due to backflow was excluded from quantification. Every 100 μmslice within 2 mm of the injection plane was imaged. The area ofdistribution on each slice was summated to calculate the total volume ofnanoparticle distribution. The same process was followed for theanalysis of the distribution of transgene expression mediated by PEIbased gene vectors encoding for eGFP.

Antibodies and Western Blotting

For western blot analysis of in vivo transfection following CED, theantibodies used included anti-GFP (B-2): sc-9996 andanti-β-actin:sc-47778 (Santa Cruz Biotechnology, Santa Cruz, Calif.).Brain tissues were lysed using brief sonication in ice PBS buffer (1 mMPMSF, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin A).Sampling buffer (10% glycerol, 2% SDS, 62.5 mM Tris-HCl, 2%β-mercaptoethanol, pH 6.8) was added and samples were boiled at 100° C.for 10 min. Samples were resorved by SDS—polyacrylamide gelelectrophoresis (PAGE) and and proteins on gels were transferred tonitrocellulose (Bio-Rad, Hercules, Calif.) using a semidry blotter(Bio-Rad, Hercules, Calif.). The membrane was blocked with 3% BSA inTBST (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.5% TWEEN-20) and incubatedovernight at 4° C. with primary antibodies Immunoblots were visualizedby enhanced chemiluminescence method. Quantification of western blotresults was performed using the Multi Gauge program (Fujifilm, Tokyo,Japan) [Elliott, et al., Mol Biol Cell, 2005. 16(2): 891-901].

Results

Whether the dense PEG coating of BPN contributes to improveddistribution following CED was assessed. To directly compare the spatialdistribution of the gene vectors with different brain penetratingcapacities following CED, Cy5-labeled BPN and Cy3-labeled CPN wereco-infused. Densely PEGylated BPN homogeneously covered the rodentstriatum, whereas less coated CPN were confined in the injection site.Within the coronal plane of injection, BPN covered a 3-fold larger areathan CPN did (FIG. 5A) and the difference in distribution wasstatistically significant (p<0.05). Moreover, the overall volume ofdistribution of BPN was calculated to be 3.1-fold higher than for CPN(FIG. 5B). The concentration of nanoparticles in the CED infusate hasbeen shown to have a significant effect on the volume of distribution[MacKay, et al. Brain Res, 2005. 1035(2): 139-53]. Indeed, co-infusinggene vectors at half the concentration resulted in 13- and 8-fold lowervolume of distribution of CPN and BPN, respectively. Even at low plasmidconcentrations BPN resulted in 4.6 fold higher volume of distribution incomparison to CPN (FIG. 21).

Given the larger area of distribution of BPN and their ability to reachcells at further distances from the point of administration, thedistribution of transfected cells following CED administration of genevectors carrying plasmid DNA encoding eGFP was assessed. UPN and CPNtreated animals demonstrated significant GFP expression surrounding theinjection site and perivascular spaces. In contrast, BPN resulted inwidespread transfection throughout the rodent striatum, which correlatedwell with the gene vector distribution analysis (FIGS. 5A-5B). Inparticular, BPN resulted in a statistically significant (p>0.05)difference in the GFP transgene expression with a 2.4- and 3.2-foldhigher volume of transfection compared to CPN and UPN, respectively(FIGS. 6A and 6B). Absolute transgene GFP expression mediated by CED ofUPN, CPN and BPN was quantitatively determined using Western blotanalysis. BPN demonstrated a statistically significant, 2-fold higheroverall transgene expression in the striatum in comparison to CPN andUPN (FIG. 7).

In contrast to the in vitro results, CED administration of BPN resultedin double the total amount of in vivo transgene expression in comparisonto UPN and CPN, suggesting that the ability of BPN to transfect cellsover a large area of the striatum may offset and even surpass theirinferior intracellular delivery capacity

The unhindered diffusion of BPN in the brain parenchyma translates towidespread distribution when administered using CED. It should be notedthat a high density surface-shielding is required to achieve aCED-facilitated distribution of gene vectors; the insufficientlyshielded CPN were unable to escape the injection site, and failed tomediate an enhanced distribution in transgene expression compared tounshielded UPN following CED.

Example 7: Synthesis of Nanoparticles Containing Poly L-Lysine andBranched PEG

Materials and Methods

Synthesis of Branched PEG (BrPEG)

BrPEG was synthesized in a two-step reaction.Diethylenetriaminepentaacetic acid (DTPA) anhydride was first conjugatedto azido-trioxaundecanin at a 1:1 molar ratio in the presence of 2 molarequivalents of N, N-Diisopropylethylamine. The azido-DTPA resulting fromthis reaction was then conjugated with 4 molar equivalents of 5 kDamethoxy-PEG-amine (Creative PEGWorks, Winston Salem, N.C.) in thepresence of 40, 5 and 3 molar equivalents of1-Ethyl-3-(3-methylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide(NHS) and 4-dimethylaminopyridine (DMAP), respectively, indimethylformamide (DMF). The reaction was carried out for 48 hours at37° C. with constant stirring. For purification reaction products weredialyzed (6-8 kDa MWCO, Spectrum Laboratories, Inc., Rancho Dominguez,Calif.) against ultrapure water for 24 hours

PEGylation of Poly-L-Lysine

Poly-L-lysine (PLL) 30-mers with functionalized alkyne end groups andbromide counterions were used (Alamanda Polymers Inc., Huntsville,Ala.). For formation of linear PEGylated PLL polymers, PLL peptides werereacted with PEG (˜5 kDa) (Creative PEGWorks, Winston Salem, N.C.) orbranched PEG (˜15 kDa) with functionalized azide groups at a molar ratioof 1:1. The click chemistry reaction was carried out at 37° C. for 48hours in the presence of 0.1 molar equivalents of copper acetate, 5molar equivalents of sodium ascorbate andTris(benzyltriazolylmethyl)amine (TBTA) in 100 mM Tris buffer (pH 7.5).Reaction products, PLL-PEG (di-block of PLL and linear PEG) andPLL-BrPEG (di-block of PLL and branched PEG), were dialyzed againstultrapure water for 24 hours.

Purification of PEGylated Peptides and Exchange of Counter-Ions

Reaction products from PEGylation reactions were purified using sizeexclusion chromatography with SEPHADEX G15 (MWCO 1500, GE healthcare,Pittsburgh, Pa.) as the stationary phase and 50 mM ammonium acetatebuffer (pH 7.4) as the mobile phase. During this process bromidecounter-ions were exchanged with acetate counter-ions. Peptideconcentrations in different fractions were monitored by measuring theabsorbance at 220 nm (NANODROP ND-1000 spectrophotometer, NANODROPTechnologies, Wilmington, Del.). High-molecular weight fractionscontaining peptide were pooled and dialyzed. Dialysis was carried outusing dialysis tubes with a MWCO of 500 Da, against 2 liters ofultrapure water which was intermittently changed over the course of 24hours. Purified products were lyophilized and stored at −80° C. untilfurther use.

Particle Formulation

Gene vectors that mimicked the conventionally PEGylated Copernicusformulation (PLL-PEG) were formulated at a nitrogen (contributed by PLL)to phosphorus (contributed by DNA) ratio of 2. To determine the mostsuitable formulation parameters for compacting DNA in densely PEGylatedgene vectors, nitrogen to phosphorus (N/P) ratios of 2 and 5 were testedusing different ratios of PLL-BrPEG and PLL polymers. Gene vectors werecomplexed using 100% PLL-BrPEG (BR 100), a mixture of either 90%PLL-BrPEG and 10% PLL (BR 90) or 50% PLL-BrPEG and 50% PLL (BR 50). Genevectors were formed by the drop-wise addition of 10 volumes of plasmidDNA (0.2 mg/ml) to 1 volume of polymer solution while vortexing at aslow speed. The plasmid/polymer solutions were incubated for 30 min atroom temperature. Syringe filtration (0.2 μm) was used for removal ofaggregates followed by removal of free polymer and collection of genevectors at desired concentration by AMICON® Ultra Centrifugal Filters(100,000 MWCO, Millipore Corp., Billerica, Mass.). DNA concentration wasdetermined via absorbance at 260 nm using a NANODROP ND-1000spectrophotometer (Nanodrop Technologies, Wilmington, Del.).

Results

Polymer Synthesis

Branched PEG (BrPEG) was synthesized and analyzed by ¹H-NMR to determinethe ratio of PEG to DTPA. The results of the NMR indicated that anaverage of 3.65 PEG molecules were attached to each DTPA molecule.PLL-PEG and PLL-BrPEG polymers were synthesized as described in thematerials and methods through click chemistry reactions (FIG. 9). The¹H-NMR spectra for polymers was used to determine the degree ofPEGylation. The ratio of PEG to peptide was calculated based on theexpected chemical shift contributed by protons attached to individualatoms. In the lysine monomer of PLL, the intensity contributed at aparticular chemical shift by protons attached to each carbon are in thefollowing ratio a:b:c:d:e=1:2:2:2:2. For carbons b and d, thecorresponding peaks were too close to be clearly distinguished and thuswere considered as a single peak. Since each lysine monomer was repeated30 times, the peaks for a, c, b+d and e would represent 30, 60, 120 and60 protons each. When keeping these as reference peaks each PEG moleculewould be expected to contribute 456 protons since each repeating unit inPEG has 4 protons and 5 kDa PEG contains 114 repeating units. Thus theresultant products revealed a calculated PEG to peptide ratio of 1.12for PLL-PEG and 3.16 for PLL-BrPEG polymers. Based on the NMR spectraboth PEGylated peptides were deemed suitable for particle formulationwith the degree of PEGylation for the densely PEGylated polymer beingapproximately 3.1 fold of the conventionally PEGylated polymer.

Particle Characterization

A comparison of TEM images revealed that conventional PLL-PEG genevectors were rod shaped while both un-PEGylated PLL gene vectors anddensely PEGylated gene vectors consisted of spherical and ellipsoidalparticles. Un-PEGylated PLL gene vectors had an average major diameterof 82 nm and an average minor diameter of 46 nm. Analysis of PLL-PEGgene vector sizes (Table 3) revealed a major diameter of 177±8 nm and aminor diameter of 16±1 nm. The major diameters of densely PEGylated genevectors were ˜2 fold smaller than PLL-PEG gene vectors, while the minordiameters were ˜2 fold larger. PLL-BrPEG gene vectors formed at an N/Pratio of 5 had significantly lower major and minor diameters incomparison to gene vectors formed at an N/P ratio of 2. Gene vectoraspect ratios, calculated using the major and minor diameters, werefound to be 1-4 fold lower for particles formed incorporating thePLL-BrPEG polymer (FIG. 10, Table 3). The range of aspect ratios wasalso lower in the case of densely PEGylated particles which wasreflected in the lower polydispersity observed for gene vectorsincorporating the PLL-BrPEG polymer (FIG. 10, Table 3). Addition ofun-PEGylated PLL generated particles with significantly smaller aspectratios (FIG. 10).

TABLE 3 Physicochemical characterization of gene vectors. Hydrodynamicdiameter Major diameter Minor diameter Aspect Zeta Potential (nm) ± SEM(nm) ± SEM (nm) ± SEM Ratio ± SEM (mV) ± SEM PDI PLL-PEG 171 ± 5 177 ±8  16 ± 1 12.0 ± 0.5  1.5 ± 0.6 0.31 (N/P 2) PLL  108 ± 13 82 ± 6 46 ± 32.4 ± 0.4 10.0 ± 1.2  0.21 (N/P 2) BR 50 135 ± 6 95 ± 3 43 ± 3 3.2 ± 0.11.1 ± 0.7 0.26 (N/P 2) BR 90 127 ± 3 87 ± 6 34 ± 2 4.0 ± 0.5 2.2 ± 1.10.20 (N/P 2) BR 100 142 ± 5 117 ± 1  32 ± 2 6.1 ± 0.4 2.2 ± 0.9 0.28(N/P 2) BR 50 119 ± 8 90 ± 3 42 ± 3 2.9 ± 0.2 2.8 ± 0.2 0.24 (N/P 5) BR90 129 ± 6 77 ± 2   32 ± 2.4 3.2 ± 0.1 1.4 ± 0.4 0.24 (N/P 5) BR 100 125± 4 89 ± 6 27 ± 3 4.8 ± 0.6 2.1 ± 0.4 0.24 (N/P 5) Table 3:Physicochemical characterization of gene vectors. Hydrodynamic diameter(Z average), ζ-potential and polydispersity (PDI) were measured by laserDoppler anemometry and dynamic light scattering in 1/15X PBS at pH 7.0and are represented as the arithmetic mean of 3 measurements ± standarderror (SEM), major and minor diameters were measured from TEM images forat least 100 particles each from 3 particle samples, using ImageJ, andare presented as the arithmetic mean ± SEM.

Well PEGylated nanoparticles can rapidly diffuse in the brain parenchymaprovided their diameter is small enough to allow for movement throughthe pores in the ECM. In fact, particles with dense PEG coating smallerthan 114 nm exhibited rapid diffusion in the ECM [4]. For this reasonwhich particle formulations would have a majority of the population witha size below this cut-off were examined. Only 29% PLL-PEG gene vectorshad a major diameter below 114 nm (FIG. 11). To study the effect ofusing BrPEG in gene vector formulations, the distribution of majordiameters of particle populations formulated at an N/P ratio of 2 werecompared. Blending of PLL-BrPEG and un-PEGylated PLL polymers at a ratioof 90:10 (BR 90) resulted in a population where ˜80% of the particleshad a major diameter below 114 nm. This was significantly higher thanthe percentage of particles below this cut-off for the BR 100 and BR50-50 formulations, ˜59% and ˜73% respectively. Although the minordiameters for all the densely PEGylated formulations were larger thanthe minor diameter of conventional PLL-PEG gene vectors, they were stillbelow the designated cut-off and would therefore not be expected tonegatively impact gene vector diffusion.

Given that the particles formulated here are not spherical in nature,the hydrodynamic diameter, defined as the diameter of a sphere that hasthe equivalent translational diffusion coefficient as the particle, is auseful parameter to describe and compare diffusivity characteristics.The hydrodynamic diameter, measured as the Z average, was found to belower for all formulations formed using the densely PEGylated polymer(Table 3). The surface charge is also an important aspect affectingdiffusion since highly charged particles are more likely toelectrostatically interact with the ECM. No significant differences werenoted in surface charge between the PEGylated particle formulations. Thesurface charge of un-PEGylated PLL gene vectors was ˜5 fold higher thanthe PEGylated formulations.

Non-specific interactions of conventional nanoparticles with componentsof the ECM impede nanoparticle distribution in the brain parenchyma,thus affecting their ability to reach target cells and achievetherapeutic effect. Effectively shielding the nanoparticle surface witha dense layer of PEG minimizes these adhesive interactions and allowsfor efficient particle penetration through the ECM. In this study, awidely used, clinically tested gene vector was used to achievewidespread and sufficiently high transgene expression in the brain. Thecomplexation parameters were analyzed for the formulation of denselycoated gene vectors and thoroughly characterized them to investigatetheir applicability for subsequent in vitro and ex vivo evaluation.

PEGylation of PLL with a single, linear PEG and multi-arm branched PEGwas achieved and verified by NMR. PLL-PEG polymers were found to complexDNA to form conventionally PEGylated gene vectors that satisfactorilymimicked the clinically tested system. Modification of PLL polymersthrough PEG grafting has previously been reported to form spherical genevectors with a diameter ˜100 nm. The use of the PLL and branched PEGdi-block also resulted in the formation of particles with a distinctlydifferent morphology from the conventional PLL-PEG system. However inthis case, both spherical and ellipsoid gene vectors with a range ofaspect ratios were formed. Despite the steric hindrance imposed by theincreased amount of hydrophilic PEG chains that may lead to inferior DNAcomplexation, gene vector formulated with PLL-BrPEG but with theincorporation of small amounts of un-PEGylated polymer resulted in moreuniform nanoparticles with a smaller range of aspect ratios. Moreover,the inclusion of un-PEGylated polymer core lead to the formulation of alarge population of nanoparticles with major diameter lower than 114 nm,thereby minimizing the possible steric hindrances that thesenanoparticles would encounter in the brain parenchyma. However, themovement of non-spherical particle is influenced by a number ofparameters and cannot be easily predicted as particles may align ortumble in the presence of flow and therefore their behavior must beexperimentally characterized

Example 8: Characterization of Nanoparticles Containing Poly L-Lysineand Branched PEG In Vitro

Materials and Methods

Cell Culture

9 L rat Gliosarcoma cells were provided by Dr. Henry Brem and werecultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen Corp.,Carlsbad, Calif.) supplemented with 1% penicillin/streptomycin(pen/strep, Invitrogen Corp., Carlsbad, Calif.) and 10% heat inactivatedfetal bovine serum (FBS, Invitrogen Corp., Carlsbad, Calif.). Rat brainprimary astrocytes were provided by Dr. Arun Venkatesan. Rat brainprimary mixed cultures were isolated form neonatal P3-P6 rats andastrocytes were isolated with the conventional shake off method.

Cells were cultured in DMEM/F12 (Invitrogen Corp., Carlsbad, Calif.)supplemented with 10% FBS and 1% penicillin-streptomycin. As per assayrequirements, cells were trypsinized by incubating with 0.25% TrypsinEDTA (Corning Inc., Tewksbury, Mass.) for 5 minutes at 37° C. followedby neutralization with media and were seeded in 96-well or 24-wellplates and allowed to adhere overnight.

Cell Uptake

For cellular uptake studies, cells were seeded at a density of 50,000cells per well in 24-well plates, and were treated for 3 hours at 37° C.with 1 μg DNA/well in its compacted nanoparticle form for the differentparticle formulations. To enable fluorescence sorting by the BD AccuriC6 Flow Cytometer, Cy3 labeled DNA was used for nanoparticle synthesis.The use of labeled DNA did not affect the formation of DNA nanoparticlesas confirmed by TEM and ζ-potential. For flow cytometry, the cells weretreated briefly with 0.04% trypan blue to quench extracellularflorescence, washed in PBS three times and then trypsinized. The trypsinwas neutralized and the cells were collected by spinning down at 1000rcf for 10 minutes. The cell pellet obtained was resuspended in 100 μlof 10% FBS in PBS and kept on ice until the samples were processed.Nanoparticle cell uptake was measured using the Accuri C6 flow cytometer(BD Biosciences, USA) with a 488 nm laser and an FL2 band-pass filterwith emission detection wavelength of 585/40 nm. Data were analyzedusing the BD Accuri C6 software. Thresholds were determined usinguntreated samples and gene vector cell uptake was compared to freeplasmid.

Luciferase Assay

Cells were seeded at a density of 50,000 cells per well in a 24-wellplate and treated with 1 mg/ml compacted pBal DNA in DMEM supplementedwith 10% FBS and 1% penicillin-streptomycin for 3 hours at 37° C. Cellswere then cultured in fresh media as normal for 3 days before beingassayed for luciferase expression. For luciferase extraction the mediawas removed and the cells were washed twice with 1×PBS. 500 μl of 1×Reporter Lysis Buffer (Promega, Madison, Wis.) was added to each welland incubated for 10 minutes at room temperature. The cells and bufferfrom each well were transferred to separate microcentrifuge tubes andsubjected to three freeze-thaw cycles and then spun down at 1,000 rpmfor 5 minutes. The supernatant was transferred into a newmicrocentrifuge and assayed immediately for luciferase activity. 100 μlof the luciferase substrate-assay buffer mixture (Promega, Madison,Wis.) was added to a polystyrene tube followed by 20 μl of thesupernatant and the luminescence was measured immediately using a 20/20nluminometer (Turner Biosystems, Sunnyvale, Calif.). The relative lightunit (RLU) was normalized with the total protein concentration measuredby Micro-BCA protein assay (Pierce Protein Biology Products, Rockford,Ill.).

Toxicity Assay

In vitro cytotoxicity of gene vectors was measured using the CellCounting kit-8 supplied by Dojindo Molecular Technologies Inc,Rockville, Md. 10,000 cells/well were seeded in 96-well plates andtreated with 1, 5, 10, 50 and 100 μg/ml compacted DNA for 24 hours inDMEM. The media was then replaced with 100 μl DMEM containing 10% FBSand 1% penicillin-streptomycin and 20 μl of Cell Counting kit-8 reagentwas added and incubated for 2 hours. Results were measuredspectrophotometrically at 450 nm using the Synergy Mx Multi-ModeMicroplate Reader (Biotek, Instruments Inc.).

Results

Toxicity

Cell viability assays on 9 L cells revealed increased toxicity ofparticles formulated at high ratios of polymer to DNA (N/P 5) comparedto their low N/P ratio counterparts (FIG. 12). The most toxicformulation was the densely PEGylated gene vector, BR 90, formed at anN/P ratio of 5. However cell viability assays for all particles at anN/P ratio of 2 for 9 L cells (FIG. 12) revealed that gene vectors wererelatively non-toxic even at concentrations as high as 100 μg/ml, ˜75%viability for all formulations tested. Further cell viability studiesrevealed that the toxicity of gene vectors formulated at N/P 2 was lowat concentrations up to 10 μg/ml for both 9 L cells and rat primaryastrocytes (FIGS. 13A and 13B).

Cell Uptake

Flow cytometric analysis showed increased uptake in the order of PLL>BR90>BR 100>PLL-PEG for the different formulations (FIGS. 14A and 14B)with differences between particles being significant (p value<0.05). Thesame trend was observed for 9 L tumor cells and primary rat astrocytes.However absolute values indicated that overall gene vector uptake washigher for 9 L cells. The uptake of PLL-PEG was close to levels observedfor free plasmid in both cell lines.

Transfection

Luciferase activity, measured in Relative Luminescence Units (RLU)(FIGS. 14C and 14D) in both 9 L cells and primary astrocytes was highestfor the BR 90, followed by the BR 100, PLL and finally PLL-PEGformulations. Differences were found to be significant only for primaryastrocytes where transfection levels for BR 90 were significantly higherthan those for all other formulations tested. Transfection of PLL-PEGwas comparable to levels achieved by free plasmid.

The toxicity of cationic polymers has been one of the most prominentfactors limiting their clinical applicability. It is therefore importantto ensure low levels of toxicity for any proposed gene vector platform.The conventional PLL-PEG formulation tested in clinical trials was foundto be non-toxic. Low in-vitro toxicity of PLL-PEG at an N/P of 2verified its applicability as a safely administrable gene vector.PLL-PEG gene vectors were found to be toxic when the N/P ratio wasincreased to 5 and this result was replicated for the rest of theformulations tested. Previous reports in literature have also indicatedan increased toxicity of PLL based gene vectors with increasing N/Pratios. As a result of the high toxicity of gene vectors observed at N/P5, only the gene vectors formulated at N/P 2 were further characterized.Additional cell viability assays conducted on 9 L and primary cellsdemonstrated low gene vector toxicity even at the highest treatmentconcentrations tested and confirmed that gene vectors were not toxic foreither cell line at concentrations used for subsequent in-vitro studies.

In-vitro characterization of gene vector internalization revealed thatcellular uptake increased as the aspect ratio decreased, with PLL-PEG(average aspect ratio of 12) and PLL (average aspect ratio 2) genevectors exhibiting the lowest and highest cellular uptake respectively.The observation is in line with previous experimental studies that haveshown particles with low aspect ratios to exhibit more rapid cellinternalization. Specifically, gold nanospheres with diameters of 14 nmor 74 nm have been shown to be taken up by HeLa cells more than threetimes as often as 74 14 nm rods (aspect ratio 5). Similarly higheraspect ratios have demonstrated reduced cellular uptake of polymer basedgene vectors formed using N-(2-hydroxypropyl) methacrylamide(HMPA)-oligolysine brush polymers. While acknowledging these findings,it is difficult to draw comparisons between such experimental studiesdue to variations in particle chemistry and cell lines tested.Theoretical models based on energy considerations have also proposedthat particles with low aspect ratios are associated with minimumwrapping time, whereas particles lying parallel to the cell are onlypartially wrapped if too elongated (“frustrated endocytosis”). Althoughreduced uptake of particles with high aspect ratios has beendemonstrated, comparisons between spherical and ellipsoidalnanoparticles have shown that a slightly elongated shape may in factimprove cell uptake. Employing a top-down approach called PRINT, Grattonet al. have demonstrated that internalization of cylindrical particlesof cross-linked PEG-based hydrogels with an aspect ratio of three wasabout four times as fast as their spherical counterparts of the samevolume. Similar aspect ratios were observed for the densely PEGylatedformulations and conferred a significant advantage in cellular uptakeover the conventional PLL-PEG gene vectors for the cell lines studied.The lower aspect ratios achieved while still maintaining non-sphericalcharacter appear to be ideal for gene vector internalization.

Despite high cellular uptake, PLL did not exhibit higher transfectionefficiencies as compared to BR 100 and BR 90. This may be due to severalintracellular factors, including lysosomal degradation, diffusionalconstraints and metabolic degradation in the cytoplasm. The denselyPEGylated BR 90 formulation exhibited the highest transfectionefficiency in both 9 L cells and rat primary astrocytes presumably due acombined effect of high cell uptake followed by efficient intracellulartrafficking to the nucleus.

Example 9: Nanoparticles Containing Poly L-Lysine and Branched PEG haveHigh Diffusivity in Brain Tissue Ex Vivo and In Vivo

Materials and Methods

Particle Stability in aCSF

Gene vector stability was assessed by incubating in aCSF (HarvardApparatus, MA) at 37° C. and recording Z-average and polydispersity(PDI) by dynamic light scattering before treatment and 0 hours and 1hour after treatment using Zetasizer Nano ZS90 (Malvern Instruments,Southborough, Mass.). Measurements were performed at 25° C. at ascattering angle of 90°. TEM girds were also prepared as discussed insection 2.4.2.2 at different time-points including pre-incubation,immediately post-incubation (0 hour), 1 hour post-incubation and 24hours post incubation.

Preparation of Rodent Brain Slices

All animal experiments were carried out at Johns Hopkins UniversitySchool of Medicine following National Institutes of Health guidelinesand local Institutional Animal Care and Use Committee regulations.Briefly, adult Fisher rats (120-140 g) were euthanized with overdose ofisoflurane and their brains rapidly removed and immersed in cold aCSF(Harvard Apparatus, MA) supplemented with 10% glucose for 10 minutes.The brain were then sliced to 1.5 mm thick slices using a Zivic brainmatrix slicer (Zivic Instruments, Pittsburgh, Pa.) and placed on custommade slides. Half a microliter of fluorescently labeled gene vectors wasinjected on the cerebral cortex at a depth of 1 mm using a 50 ulHamilton Neuro Syringe (Hamilton, Reno, Nev.) mounted on a stereotaxicframe. Tissues were covered by a 22 mm×22 mm coverslip to reduce tissuemovement and bulk flow.

Multiple Particle Tracking

Particle trajectories were recorded over 20 seconds at an exposure timeof 66.7 ms by a Evolve 512 EMCCD camera (Photometrics, Tucson, Ariz.)mounted on an inverted epifluorescence microscope (Axio Observer D1,Zeiss; Thornwood, N.Y.) equipped with a 100×/1.46 NA oil-immersionobjective. Movies were analyzed by extracting x, y-coordinates of genevectors centroids over time and the mean square displacement of eachparticle was calculated as a function of time. At least N=3 rat brainswere used per gene vector type and at least 500 gene vectors weretracked per sample. The geometric mean of the MSDs for all nanoparticleswas calculated per sample and the average of different rodent brains wascalculated as a function of time. Histograms were generated from the MSDof every nanoparticle at a time scale of τ=1 sec

In Vivo Co-Injection

To study nanoparticle diffusion in vivo, equal concentrations ofdifferentially fluorescently labeled PLL-PEG and BR 90 nanoparticleswere co-injected into the striatum of female Fischer 344 rats (n=3). Therodents were anesthetized with a mixture of ketamine-xylazine and amid-sagittal incision was made to expose the bregma. The striatum wastargeted 0.5 mm posterior to the bregma and 3 mm lateral to the midline.The nanoparticle loaded 50 μl Hamilton Neuros syringe was lowered 3.5 mmbelow the dura. Gene vectors at individual concentrations of 0.25 μg/μlwere administered at a rate of 0.33 μl/min, using a Chemyx Inc. NanojetStereotaxic syringe pump (Chemyx, Stafford, Tex.), for a total of 20 μlfollowed by withdrawal of the syringe. The animal was sutured and placedon a heating pad before returning to its cage. After 2 hours the rodentwas sacrificed, the brain was removed, and fixed in formalin overnight.The suspension solution was then changed to 15% sucrose and subsequentlyto 30% sucrose after 24 hours. The brain was sliced using a Leica CM1905 cryostat to obtain slices of 100 μm thickness. The slices werestained with DAPI (Molecular Probes, Eugene, Oreg.) and imaged using theZeiss LSM 510 Meta Confocal Microscope at 5× magnification (Carl Zeiss;Hertfordshire, UK). Microscope settings were kept constant duringimaging. Brain slice images were quantified for fluorescent distributionof BR 90 or PLL-PEG nanoparticles by running the confocal laser scanningmicroscope images through a custom MATLAB script which thresholded theimages at 10% of the maximum intensity. Care was taken to avoidquantifying fluorescent distribution in the ventricles or white mattertracts. The area of distribution calculated from each slice wasmultiplied by the slice thickness of 100 μm and summated across allimages to obtain a total volume of distribution.

Results

Particle Stability in aCSF

Gene vector stability was investigated in aCSF to model thephysicochemical characteristics of the gene vectors following injectioninto the brain. The PLL-PEG formulation was unstable immediately uponimmersion in CSF as both Z-average and the polydispersity index (PDI)increased significantly when compared to particle characteristicsmeasured prior to treatment (FIG. 15). At one hour after treatment, thePDI further increased while a drop in the Z average was registered. Forboth the highly PEGylated formulations, BR 100 and BR 90, a slightincrease in Z-average and PDI was observed immediately after CSFtreatment. In the case of BR 100, both measured values dropped at theone hour time-point where as the Z-average and PDI maintained at thesame levels for BR 90. These results were corroborated with TEM imagestaken for each sample at the same time-points.

Particle Diffusion

Automated tracking of gene vectors was used to obtain the ensemblegeometric mean square displacement up to a timescale of τ=1 second foreach tissue sample and was then averaged over three different samples.The densely PEGylated gene vectors showed improved transport over theconventional PLL-PEG (FIG. 16). The MSDs at τ=1 second for BR 90 and BR100 were 5.6 and 4.2 fold higher than that of the conventional PLL-PEGformulation. The differences between BR 90 and BR 100 were not found tobe significant. The increased log(MSD) for the densely PEGylatednanoparticles can also be seen in the histogram plots (FIG. 18). Whenanalyzing individual gene vector MSDs, a greater percentage of particleswith log 10MSD≥−1, defined as rapidly moving, were observed for BR 90(58.8%) and BR 100 (46.3%) versus PLL-PEG (28.2%). Representativeparticle trajectories (FIG. 17) depict the poor diffusivity of PLL-PEGwhile BR 90 and BR 100 were diffusive over large distances. PLLparticles were completely immobilized.

In Vivo Co-Injections

In vivo co-injections of gene vectors revealed that densely PEGylated BR90 particles were able to distribute further than the conventionallyPEGylated PLL particles. Quantitative analysis of confocal images showedthat the areas of distribution followed a bell shaped curve (FIG. 19),with maximum distribution in the plane of the site of injection. Thevolume of distribution for the densely PEGylated gene vector wasapproximately four fold higher than that for PLL-PEG (FIG. 20).

The concurrent improvement in brain penetration and in vitrotransfection efficiency achieved with DNA-BPN in comparison to DNA-CPNled to enhanced and more widespread transgene expression in vivo, asdemonstrated with CED. Quantitative analysis of the integrated density(FIG. 22A) and number of cells transfected per brain slice at distancesup to 500 μm away from the injection plane, revealed a significant,approximately 2-3 fold increase, for BR 90 (N/P2) particles (DNA-BPN) incomparison to PLL-PEG particles (DNA-CPN) (FIG. 22B).

PLL-PEG exhibited limited diffusion in the brain parenchyma. Incontrast, densely PEGylated PLL gene vectors exhibit significantlyimproved diffusivities in ex vivo and in vivo rodent brain tissue ascompared to the conventional PLL-PEG system. Among the densely PEGylatedformulations, the marginally higher diffusivity of BR 90 compared to BR100, could potentially be reflecting subtle differences inphysicochemical characteristics. For densely PEGylated nanoparticlesadhesive interactions with the ECM are drastically minimized andtherefore steric hindrances may play a major role. Densely PEGylatedparticles up to 114 nm are capable of brain penetration while particlesof 200 nm are not. Thus, 114 nm is a size cut-off under which denselyPEGylated particles can be expected to rapidly diffuse. Theincorporation of un-PEGylated polymer to the BR 100 gene vectorsresulted in a greater percentage of the population with a major diameteri.e. the larger dimension for non-spherical particles, below 114 nm.This, along with a lower PDI, may have contributed to a larger diffusivefraction of the BR 90 formulation. The increased brain penetratingability of densely PEGylated PLL gene vectors is a significantimprovement over the current widely used PLL-PEG gene vector. Incomparison, the particle formulation demonstrates higher stability, invitro transfection efficiency and diffusivity in the brain ECM,establishing it as a promising platform for gene delivery to the brain.

The PLL based gene vector formulated using a PLL-branched PEG co-polymerand a small amount of un-PEGylated PLL polymer demonstrated bothimproved diffusion and transfection in comparison to the conventionallyused PLL-PEG. Through dense PEGylation, charge based interactions thatlimit gene vector distribution in the brain parenchyma were minimized,while simultaneously achieving small nanoparticle sizes to minimizesteric hindrances and enhancing the volume of distribution through CED.In addition to maximizing therapeutic distribution, the physicochemicalcharacteristics of the densely PEGylated gene vector system also favoredimproved in vitro cell uptake and transfection. Moreover, the PLL-PEGsystem has demonstrated a favorable safety profile in clinical trialsfor treatment of CF, thereby setting an excellent precedence for alsotranslating a densely PEGylated PLL based gene vector system. Thecombination of rapid diffusion and efficient transgene delivery with apressure driven administration method opens a window of opportunity foreffective non-viral gene therapy of GB.

We claim:
 1. A method for delivering an effective amount of therapeutic,prophylactic or diagnostic nucleic acids through the blood brain barrierand through the anisotropic and electrostatically charged extracellularmatrix of the brain of a subject, comprising administering to thesubject a formulation comprising nanoparticles formed of a cationicfirst polymer; and a hydrophilic second polymer selected from the groupconsisting of a polyethylene glycol and copolymers thereof, wherein atleast 50% up to 90% of the cationic first polymer is conjugated to thehydrophilic second polymer, wherein the first cationic polymer, thehydrophilic second polymer, or both, are branched; and the nucleicacids, wherein the nucleic acids are encapsulated within thenanoparticles or associated with the surface of the nanoparticles, andwherein the nanoparticles have a diameter of between about 40 nm andabout 150 nm and are coated with the hydrophilic second polymer at adensity that imparts a near neutral charge and enhances diffusivitythrough the blood brain barrier and through the anisotropic andelectrostatically charged extracellular matrix of the brain.
 2. Themethod of claim 1, wherein the formulation is administered directly tothe brain.
 3. The method of claim 1, wherein the formulation isadministered systemically.
 4. The method of claim 1, wherein theformulation is administered in combination with one or more techniquesto facilitate passage of the nanoparticles through the blood brainbarrier.
 5. The method of claim 4, wherein the technique is selectedfrom the group consisting of convection enhanced delivery, electronparamagnetic resonance, ultrasound sonication with or withoutmicrobubbles, and use of osmotic agents.
 6. The method of claim 1,wherein the subject has one or more symptoms of a disease or disorderselected from the group consisting of tumors, neurological disorders,and brain injury or trauma.
 7. The method of claim 1, wherein more than32.9% of the nanoparticles have an ensemble geometric mean squaredisplacement of at least 0.1 μm² at a timescale of 1 second in thebrain.
 8. The method of claim 1, wherein the nanoparticles have adiameter of less than or equal to 114 nm, 100 nm, or 50 nm.
 9. Themethod of claim 1, wherein the hydrophilic second polymer ispolyethylene glycol having a molecular weight between 1,000 Daltons and10,000 Daltons.
 10. The method of claim 9, wherein the polyethyleneglycol has a molecular weight of 5,000 Daltons.
 11. The method of claim1, wherein the cationic first polymer is branched polyethyleneimine witha molecular weight between 10,000 Daltons and 50,000 Daltons.
 12. Themethod of claim 1, wherein the molar ratio of the hydrophilic secondpolymer to the cationic first polymer is greater than
 8. 13. The methodof claim 1, wherein the nitrogen-to-phosphorus ratio of the cationicfirst polymer and the hydrophilic second polymer to the nucleic acids isat least
 2. 14. The method of claim 1, wherein the cationic firstpolymer is poly-L-lysine.
 15. The method of claim 14, wherein thehydrophilic second polymer is branched polyethylene glycol.
 16. Themethod of claim 15, wherein at least 50% of the poly-L-lysine isconjugated with the branched polyethylene glycol.
 17. The method ofclaim 1, wherein the mass of the hydrophilic second polymer is at least1/10,000, 1/7500, 1/5000, 1/4000, 1/3400, 1/2500, 1/2000, 1/1500,1/1000, 1/500, 1/250, 1/200, 1/150, 1/100, 1/75, 1/50, 1/25, 1/20, 1/5,1/2, or 9/10 of the mass of the nanoparticles.
 18. The method of claim1, wherein the weight percent of the hydrophilic second polymer relativeto total nanoparticle is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or greater.
 19. The methodof claim 1, wherein the formulation further comprises a pharmaceuticallyacceptable excipient.
 20. The method of claim 5, wherein the osmoticagents comprise mannitol.